WO2023006975A2 - Protéines chimériques et méthodes d'immunothérapie - Google Patents

Protéines chimériques et méthodes d'immunothérapie Download PDF

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WO2023006975A2
WO2023006975A2 PCT/EP2022/071407 EP2022071407W WO2023006975A2 WO 2023006975 A2 WO2023006975 A2 WO 2023006975A2 EP 2022071407 W EP2022071407 W EP 2022071407W WO 2023006975 A2 WO2023006975 A2 WO 2023006975A2
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cells
chimeric protein
terminus
trimeric
signaling
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PCT/EP2022/071407
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WO2023006975A3 (fr
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Nicolas BOISGERAULT
Tacien PETITHOMME
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Nantes Université
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Priority to US18/291,271 priority Critical patent/US20240279336A1/en
Priority to CN202280053156.9A priority patent/CN117813320A/zh
Priority to EP22757588.3A priority patent/EP4377333A2/fr
Priority to KR1020247006122A priority patent/KR20240038768A/ko
Priority to JP2024505279A priority patent/JP2024529484A/ja
Publication of WO2023006975A2 publication Critical patent/WO2023006975A2/fr
Publication of WO2023006975A3 publication Critical patent/WO2023006975A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20211—Vesiculovirus, e.g. vesicular stomatitis Indiana virus
    • C12N2760/20241—Use of virus, viral particle or viral elements as a vector
    • C12N2760/20243—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention is in the field of immunotherapy. More particularly, the invention relates to methods and compositions for selecting a cancer treatment in a subject suffering from cancer.
  • a fusion protein is an artificial protein obtained by the combination of different proteins, or parts of proteins. It is obtained following the creation by recombination of DNA of a gene comprising the open reading frames corresponding to the desired proteins or parts of proteins. Fusion proteins can also be called chimeric proteins.
  • Classical therapeutic proteins for cancer immunotherapy target immunomodulatory proteins located on the surface of both tumour cells and immune cells for either agonistic or antagonistic activity.
  • Chimeric proteins for cancer immunotherapy can combine these activities by displaying both agonistic and antagonistic properties on a single molecule. However, those usually exhibit toxicities related to their potency being exerted in the entire organism and not only in tissues relevant for cancer treatment.
  • This may be solved by using appropriate vectorization of the chimeric proteins to the tumour microenvironment, where immune cells interact with tumour cells expressing immunomodulatory molecules. This vectorization can be achieved by inserting the genes coding for the chimeric proteins in gene therapy vectors, including viral vectors such as oncolytic viruses that specifically replicate in tumours.
  • the present invention relates to methods and compositions for selecting a cancer treatment in a subject suffering from cancer.
  • the present invention is defined by the claims.
  • the inventors built novel immunotherapeutic molecules to specifically and precisely reverse cell-targeted immune responses in different pathological contexts (e.g. cancer, transplantation, allergy, autoimmune diseases).
  • pathological contexts e.g. cancer, transplantation, allergy, autoimmune diseases.
  • the present injection relates to a nucleic acid encoding a chimeric protein having a general structure of:
  • the present invention relates to a nucleic acid encoding a chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus wherein (a) is a domain, (b) is a functional linker and (c) is a domain.
  • the present invention relates to a nucleic acid encoding a chimeric protein having a general structure of: N terminus - (c) - (b) - (a) - C terminus wherein (a) is a domain, (b) is a functional linker and (c) is a domain.
  • the present invention relates to a nucleic acid encoding a chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus wherein (a) is a signaling and/or targeting domain, (b) is a functional linker and (c) is a signaling and/or targeting domain.
  • the present invention relates to a nucleic acid encoding a chimeric protein having a general structure of: N terminus - (c) - (b) - (a) - C terminus wherein (a) is a signaling and/or targeting domain, (b) is a functional linker and (c) is a signaling and/or targeting domain.
  • the present invention relates to a nucleic acid encoding a chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus wherein (a) is a monomeric domain, (b) is a functional linker and (c) is a trimeric (e.g. homotrimeric or heterotrimeric) domain.
  • the present invention relates to a nucleic acid encoding a chimeric protein having a general structure of: N terminus - (c) - (b) - (a) - C terminus wherein (c) is a monomeric domain, (b) is a functional linker and (a) is a trimeric (e.g. homotrimeric or heterotrimeric) domain.
  • the present invention relates to a nucleic acid encoding a chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus wherein (a) is a monomeric domain, (b) is a functional linker and (c) is a dimeric (e.g. homodimeric, heterodimeric) domain.
  • the present invention relates to a nucleic acid encoding a chimeric protein having a general structure of: N terminus - (c) - (b) - (a) - C terminus wherein (c) is a monomeric domain, (b) is a functional linker and (a) is a dimeric (e.g. homodimeric, heterodimeric) domain.
  • the present injection relates to a chimeric protein having a general structure of:
  • the present invention relates to a chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus wherein (a) is a domain, (b) is a functional linker and (c) is a domain.
  • the present invention relates to a chimeric protein having a general structure of: N terminus - (c) - (b) - (a) - C terminus wherein (a) is a domain, (b) is a functional linker and (c) is a domain.
  • the present invention relates to a chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus wherein (a) is a signaling and/or targeting domain, (b) is a functional linker and (c) is a signaling and/or targeting domain.
  • the present invention relates to a chimeric protein having a general structure of: N terminus - (c) - (b) - (a) - C terminus wherein (a) is a signaling and/or targeting domain, (b) is a functional linker and (c) is a signaling and/or targeting domain.
  • the present invention relates to a chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus wherein (a) is a targeting monomeric domain, (b) is a functional linker and (c) is a trimeric (e.g. homotrimeric or heterotrimeric) domain.
  • the present invention relates to a chimeric protein having a general structure of: N terminus - (c) - (b) - (a) - C terminus wherein (c) is a monomeric domain, (b) is a functional linker and (a) is a trimeric (e.g. homotrimeric or heterotrimeric) domain.
  • the present invention relates to a chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus wherein (a) is a monomeric domain, (b) is a functional linker and (c) is a dimeric (e.g. homodimeric, heterodimeric) domain.
  • the present invention relates to a chimeric protein having a general structure of: N terminus - (c) - (b) - (a) - C terminus wherein (c) is a monomeric domain, (b) is a functional linker and (a) is a dimeric (e.g. homodimeric, heterodimeric) domain.
  • the chimeric protein of the invention is composed of a targeting domain (a) and/or a targeting domain (c).
  • the present invention relates to a chimeric protein having a general structure of: N terminus - (a) - (c) - (b) - C terminus wherein (c) is a monomeric domain, (b) is a functional linker and (a) is a dimeric (e.g. homodimeric, heterodimeric) domain or a trimeric (e.g. homotrimeric or heterotrimeric) domain.
  • the chimeric protein of the invention has a targeting domain (a) that specifically binds to molecules at the surface of the cells to be targeted for immune destruction or protection or immune reprogramming. In some embodiments, the chimeric protein has a targeting domain (c) that specifically binds to molecules at the surface of the cells to be targeted for immune destruction or protection or immune reprogramming.
  • the chimeric protein of the invention is composed of a signaling domain (a) and/or a signaling domain (c).
  • the chimeric protein of the invention has a signaling domain (a) that initiates a signal transduction cascade. In some embodiments, the chimeric protein has a signaling domain (c) that initiates a signal transduction cascade.
  • the chimeric protein of the invention has a domain (a) which is a targeting domain and/or a signaling domain. In some embodiments, the chimeric protein of the invention has a domain (c) which is a targeting domain and/or a signaling domain.
  • chimeric protein refers to proteins created through the joining of two or more genes that originally coded for separate proteins. Translation of this fusion gene results in a single or multiple polypeptides with functional properties derived from each of the original proteins.
  • the chimeric protein comprises a first amino acid sequence linked to a second amino acid sequence with which it is not naturally linked in nature.
  • the amino acid sequences may normally exist in separate proteins that are brought together in the fusion protein or they may normally exist in the same protein but are placed in a new arrangement in the fusion protein.
  • a chimeric protein may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.
  • TAME-IT refers to the chimeric protein of the present invention.
  • the chimeric protein of the invention is capable of binding murine ligand(s)/receptor(s). In some embodiments, the chimeric protein of the invention is capable of binding human ligand(s)/receptor(s).
  • the chimeric protein is an activator protein.
  • the chimeric protein is engineered to enhance, increase, and/or stimulate the transmission of an immune stimulatory signal.
  • the chimeric protein is an inhibitor protein.
  • the chimeric protein is engineered to inhibit, decrease, and/or block the transmission of an immune stimulatory signal.
  • the chimeric protein of the invention comprises an anti-PD-Ll and is paired with a monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) immune stimulatory agent as follows: anti-PD-Ll/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) 4-1 BBL; anti-PD-Ll/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) OX-40L; anti-PD-Ll/ monomeric or dimeric (e.g.
  • homodimeric, heterodimeric) or trimeric e.g. homotrimeric or heterotrimeric LIGHT
  • anti- PD-Ll/ monomeric or dimeric e.g. homodimeric, heterodimeric
  • trimeric e.g. homotrimeric or heterotrimeric
  • anti-PD-Ll/ monomeric or dimeric e.g. homodimeric, heterodimeric
  • anti-PD-Ll/ monomeric or dimeric e.g. homodimeric, heterodimeric
  • trimeric e.g. homotrimeric or heterotrimeric
  • CD30L anti-PD-Ll/ monomeric or dimeric
  • anti-PD-Ll/ monomeric or dimeric e.g.
  • homodimeric, heterodimeric) or trimeric e.g. homotrimeric or heterotrimeric
  • CD40L anti-PD-Ll/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) HVEM, anti- PD-Ll/ monomeric or dimeric or trimeric (e.g. homotrimeric or heterotrimeric) GITR; anti-PD- LI/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) CD27; anti-PD-Ll/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g.
  • CD28 anti-PD-Ll/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) CD 137 and/or anti-PD-Ll/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) TL1 A.
  • the chimeric protein is anti-PD- Ll -Fc-LIGHT monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g.
  • the anti-PD-Ll is a nanobody or a scFv.
  • the anti- PD-Ll nanobody is KN035 (see Zhang et al. Cell Discov. 2017; 3: 17004) or 5DXW (see https://www.rcsb.org/structure/5DXW).
  • the anti- PD-L1 scFv is aPD-Ll.
  • the chimeric protein comprises anti-PD-L2 and is paired with a monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) immune stimulatory receptor as follows: anti-PD-L2/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) 4-1 BBL; anti-PD-L2/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) OX-40; anti-PD-L2/ monomeric or dimeric (e.g.
  • homodimeric, heterodimeric or trimeric (e.g. homotrimeric or heterotrimeric) CD30; anti-PD-L2/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) CD40 and anti-PD-L2/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) CD137.
  • the chimeric protein of the invention comprises an anti-TIM-3 and is paired with a monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) immune stimulatory agent as follows: anti-TIM-3/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) OX-40L; anti-TIM-3/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) LIGHT; anti-TIM-3/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g.
  • the chimeric protein is anti-TIM3- Fc-OX40L monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric), in which the Fc represents a linker that comprises at least a portion of an Fc domain of an antibody and which comprises at least one cysteine residue capable of forming a disulfide bond.
  • the chimeric protein of the invention comprises an anti-CTLA-4 and is paired with a monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) immune stimulatory agent as follows: anti-CTLA-4/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) 4-1 BBL, anti-CTLA-4/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) OX-40L; anti-CTLA-4/ monomeric or dimeric (e.g.
  • homodimeric, heterodimeric or t(e.g. homotrimeric or heterotrimeric) rimeric CD40L; anti-CTLA-4/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or (e.g. homotrimeric or heterotrimeric) trimeric CD137L; anti-CTLA-4/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or (e.g. homotrimeric or heterotrimeric) trimeric TL1A; and anti-CTLA-4/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) OX40L.
  • anti-CTLA-4/ monomeric or dimeric e.g. homodimeric, heterodimeric
  • trimeric e.g. homotrimeric or heterotrimeric
  • the anti-CTLA-4 is a scFv.
  • the anti-CTLA-4 scFv is aCTLA-4 (see Griffin et al. J Immunol 2000 May 1; 164(9): 4433 -42).
  • the chimeric protein of the invention comprises an anti-TIGIT and is paired with a monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric immune stimulatory agent as follows: anti-TIGIT/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) 4-1 BBL, anti-TIGIT/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) OX-40L; anti-TIGIT/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g.
  • homotrimeric or heterotrimeric LIGHT LIGHT
  • anti-TIGIT/ monomeric or dimeric e.g. homodimeric, heterodimeric
  • trimeric e.g. homotrimeric or heterotrimeric
  • CD30L anti-TIGIT/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g.
  • CD40L anti-TIGIT/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) CD137L; anti-TIGIT/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) TL1A; and anti-TIGIT/ monomeric or dimeric or trimeric (e.g. homotrimeric or heterotrimeric) OX40L.
  • anti-TIGIT/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) TL1A anti-TIGIT/ monomeric or dimeric or trimeric (e.g. homotrimeric or heterotrimeric) OX40L
  • the chimeric protein of the invention comprises an anti-VISTA and is paired with a monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) immune stimulatory agent as follows: anti-VISTA /4-1 BBL monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric), anti-VISTA/OX-40L monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric); anti-VISTA /LIGHT monomeric or dimeric (e.g.
  • homodimeric, heterodimeric) or trimeric e.g. homotrimeric or heterotrimeric
  • anti-VISTA/GITRL monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric)
  • anti-VISTA/CD70 monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric)
  • anti-VISTA/CD30L monomeric or dimeric e.g. homodimeric, heterodimeric
  • anti-VISTA/CD40L monomeric or dimeric (e.g.
  • homodimeric, heterodimeric) or trimeric e.g. homotrimeric or heterotrimeric
  • anti-VISTA/CD137L monomeric or dimeric e.g. homodimeric, heterodimeric
  • trimeric e.g. homotrimeric or heterotrimeric
  • anti-VISTA/TL1A monomeric or dimeric e.g. homodimeric, heterodimeric
  • trimeric e.g. homotrimeric or heterotrimeric
  • anti-VISTA/OX40L monomeric or dimeric e.g. homodimeric, heterodimeric
  • homotrimeric or heterotrimeric e.g. homotrimeric or heterotrimeric
  • the chimeric protein of the invention comprises an anti-BTLA and is paired with a monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric immune stimulatory agent as follows: anti-BTLA/4-1 BBL monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric), anti-BTLA/OX- 40L monomeric or dimeric or trimeric (e.g. homotrimeric or heterotrimeric); anti-
  • BTLA/LIGHT monomeric or dimeric e.g. homodimeric, heterodimeric
  • trimeric e.g. homotrimeric or heterotrimeric
  • anti-BTLA/GITRL trimeric e.g. homotrimeric or heterotrimeric
  • anti-BTLA/CD70 monomeric or dimeric e.g. homodimeric, heterodimeric
  • trimeric e.g. homotrimeric or heterotrimeric
  • anti-BTLA/CD30L monomeric or dimeric e.g. homodimeric, heterodimeric
  • anti-BTLA/CD30L monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric)
  • BTLA/CD40L monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric); anti -B TLA/CD 137L monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric); anti-
  • BTLA/TL1A monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric); and anti-BTLA/OX40L monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric).
  • the chimeric protein of the invention comprises an anti- CD 172a(SIRPl a) and is paired with a monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) immune stimulatory agent as follows: anti-CD 172a(SIRPl a)/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) 4-1 BBL, anti-CD 172a(SIRPl a)/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g.
  • CD30L anti-CD 172a(SIRP la)/ monomeric or dimeric or trimeric (e.g. homotrimeric or heterotrimeric) CD40L; anti-CD 172a(SIRPl a)/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) CD137L; anti-CD 172a(SIRPl a)/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) TL1A; and anti- CD 172a(SIRPl a)/ monomeric or dimeric (e.g.
  • the chimeric protein is anti- CD 172a(SIRPl a)-Fc-CD40L trimeric (e.g. homotrimeric or heterotrimeric) or anti- CD172a(SIRPla)-Fc-LIGHT monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric), in which the Fc represents a linker that comprises at least a portion of an Fc domain of an antibody and which comprises at least one cysteine residue capable of forming a disulfide bond.
  • the chimeric protein of the invention comprises an anti-CD115 and is paired with a monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) immune stimulatory agent as follows: anti-CD 115/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) OX-40L; anti-CD 115/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) LIGHT; anti-CD115/ monomeric or dimeric (e.g.
  • homodimeric, heterodimeric or trimeric (e.g. homotrimeric or heterotrimeric) TL1A; and anti- CD 115/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) OX40L.
  • the chimeric protein of the invention comprises an anti-TMIGD2 and is paired with a monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) immune stimulatory agent as follows: anti-TMIGD2/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) OX-40L; anti-TMIGD2/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) LIGHT; anti-TMIGD2/ monomeric or dimeric (e.g.
  • the chimeric protein of the invention comprises an anti-CD200 and is paired with a monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) immune stimulatory agent as follows: anti-CD200/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) OX-40L; anti-CD200/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) LIGHT; anti-CD200/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g.
  • homotrimeric or heterotrimeric GITRL anti- CD200/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) CD70; anti-CD200/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) CD30L; anti-CD200/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) CD40L; anti-CD200/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g.
  • CD137L CD137L
  • anti-CD200/ monomeric or dimeric e.g. homodimeric, heterodimeric
  • trimeric e.g. homotrimeric or heterotrimeric
  • anti-CD200/ monomeric or dimeric e.g. homodimeric, heterodimeric
  • trimeric e.g. homotrimeric or heterotrimeric
  • the chimeric protein of the invention comprises an anti-CD 19 and is paired with a monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) immune stimulatory agent as follows: anti- CD 19/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) 4-1 BBL, anti- CD19/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) OX-40L; anti- CD 19 monomeric or dimeric (e.g.
  • homodimeric, heterodimeric) or trimeric e.g. homotrimeric or heterotrimeric LIGHT
  • anti- CD ⁇ / monomeric or dimeric e.g. homodimeric, heterodimeric
  • trimeric e.g. homotrimeric or heterotrimeric
  • anti- CD 19 monomeric or dimeric e.g. homodimeric, heterodimeric
  • trimeric e.g. homotrimeric or heterotrimeric
  • CD70 anti- CD 19/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) CD30L
  • anti- CD 19/ monomeric or dimeric e.g.
  • the anti-CD 19 is a nanobody or a scFv.
  • the anti-CD 19 scFv is an aCD19 (see Ng et al. Proc Natl Acad Sci U S A. 2012 Sep 4; 109(36): 14526-31).
  • the chimeric protein of the invention comprises an anti- MSLN (i.e. anti-mesothelin) and is paired with a monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) immune stimulatory agent as follows: anti-MSLN/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) 4-1 BBL, anti-MSLN/ monomeric or dimeric (e.g. homodimeric, heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) OX-40L; anti- MSLN monomeric or dimeric(e.g.
  • homodimeric, heterodimeric) or trimeric e.g. homotrimeric or heterotrimeric LIGHT
  • anti-MSLN / monomeric or dimeric e.g. homodimeric, heterodimeric
  • trimeric e.g. homotrimeric or heterotrimeric
  • anti-MSLN monomeric or dimeric e.g. homodimeric, heterodimeric
  • trimeric e.g. homotrimeric or heterotrimeric
  • CD30L anti-MSLN/ monomeric or dimeric (e.g.
  • the anti-MSLN is a nanobody.
  • the anti-MSLN nanobody is Al (Prantner et al, J Biomed Nanotechnol. 2015 Jul; 11(7): 1201-12).
  • the chimeric protein of the invention is a human chimeric protein. In some embodiments, the chimeric protein of the invention is a murine chimeric protein. In some embodiments, the chimeric protein of the invention is a rodent chimeric protein. In some embodiments, the chimeric protein of the invention is a feline chimeric protein a mammal. In some embodiments, the chimeric protein of the invention is canine chimeric protein. In some embodiments, the chimeric protein of the invention is a primate chimeric protein. In some embodiments, the chimeric protein of the invention is a non-human (i.e. another specie other than human) chimeric protein.
  • the chimeric protein is [Anti-PD-Ll] - [TAA] n - [dimeric 4-1 BBL and monomeric OX-40L], wherein n is gratis to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the chimeric protein is [Anti-PD-Ll] - [TAA] n - [dimeric OX-40L and monomeric 4-1 BBL] wherein n is perennial to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the chimeric protein is [Anti-PD-Ll] - [TAA] n - [trimeric 4-1 BBL], wherein n is gratis to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the chimeric protein is [Anti-PD-Ll] - [TAA] n - [trimeric OX-40L], wherein n is gratis to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the chimeric protein is [Anti-MSLN] - [TAA] n - [trimeric OX-40L], wherein n is gratis to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the chimeric protein is [Anti-PD-Ll] - [TAA] n - [trimeric hCD40L], wherein n is gratis to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the chimeric protein is [Anti-MSLN] - [TAA] n - [trimeric hCD40L], wherein n is gratis to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the chimeric protein is [Anti-MSLN] - [TAA] n - [trimeric 4-1 BBL], wherein n is gratis to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the chimeric protein is [Anti-CTLA-4] - [TAA] n - [trimeric 4-1 BBL], wherein n is gratis to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the chimeric protein is [Anti-CTLA-4] - [TAA] n - [trimeric CD40L], wherein n is gratis to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the chimeric protein is [Anti-CD 19] - [TAA] n - [trimeric CD40L], wherein n is gratis to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the chimeric protein is [Anti-mPD-Ll] - [TAA] n - [trimeric 4-1 BBL], wherein n is gratis to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the chimeric protein is [Anti-mPD-Ll] - [TAA] n - [dimeric 4-1 BBL], wherein n is gratis to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the chimeric protein is [Anti-mPDl] - [TAA] n - [trimeric 4-1 BBL], wherein n is gratis to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the chimeric protein is [dimeric Anti-mPDl] - [TAA] n - [trimeric 4-1 BBL], wherein n is gratis to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the chimeric protein of the invention binds to human PD-L1 or PD-L2 with a KD of about 1 nM to about 5 nM, for example, about 1 nM, about 1.5 nM, about 2 nM, about 2.5 nM, about 3 nM, about 3.5 nM, about 4 nM, about 4.5 nM, or about 5 nM.
  • the chimeric protein binds to human PD-L1 with a KD of about 5 nM to about 15 nM, for example, about 5 nM, about 5.5 nM, about 6 nM, about 6.5 nM, about 7 nM, about 7.5 nM, about 8 nM, about 8.5 nM, about 9 nM, about 9.5 nM, about 10 nM, about 10.5 nM, about 1 1 nM, about 1 1.5 nM, about 12 nM, about 12.5 nM, about 13 nM, about 13.5 nM, about 14 nM, about 14.5 nM, or about 15 nM..
  • the chimeric protein of the invention binds to human PD-L1 or PD-L2 with a KD of about 1 nM to about 30 nM, for example about 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 21 nM, 22 nM, 23 nM,24 nM, 25 nM,26 nM, 27 nM; 28 nM, 29 nM, 30 nM.
  • the chimeric protein of the invention binds to human PD-L1 or PD-L2 with a KD of about 1 nM to about lOOOnM, for example about 1 nM, 100 nM, 200 nM,300 nM,400 nM, 500 hM,600 nM, 700 nM, 80 nM, 900 nM, 1000 nM.
  • the present chimeric proteins of the invention are capable of, and can be used in methods comprising, promoting immune activation (e.g., against tumors).
  • the present chimeric proteins are capable of, and can be used in methods comprising, suppressing immune inhibition (e.g., that allows tumors to survive).
  • the present chimeric proteins provide improved immune activation and/or improved suppression of immune inhibition due to the proximity of signaling that is provided by the chimeric nature of the constructs.
  • the present chimeric proteins are capable of, or can be used in methods comprising, modulating the amplitude of an immune response, e.g., modulating the level of effector output.
  • the present chimeric proteins alter the extent of immune stimulation as compared to immune inhibition to increase the amplitude of a T cell response, including, without limitation, stimulating increased levels of cytokine production, proliferation or target killing potential.
  • the use of trimeric signaling molecules improves the therapeutic efficacy compared to monomers.
  • the chimeric protein of the invention is composed of a targeting domain (a) and/or a targeting domain (c).
  • targeting domain refers to a domain having functions of recognition and anchoring of on the surface of specific cells in tissues or organs of interest.
  • the targeting domain of the present invention recognizes a marker on the surface of specific cells, for instance tumor cells or immune cells; this anchors and displays the TAME-IT molecule on the cell surface and may signal inside the targeted cells.
  • the targeting domain of the present invention also can block a signal by competition.
  • the chimeric protein of the invention is composed of a monomeric targeting domain (a) or a dimeric (e.g. homodimeric or heterodimeric) targeting domain (a) or trimeric (e.g. homotrimeric or heterotrimeric) targeting domain (a).
  • the chimeric protein of the invention is composed of a monomeric targeting domain (c) or a dimeric (e.g. homodimeric or heterodimeric) targeting domain (c) or trimeric (e.g. homotrimeric or heterotrimeric) targeting domain (c).
  • the targeting domain (a) or the targeting domain (c) specifically binds to molecules at the surface of the cells to be targeted for immune destruction (e.g. tumor cells) or protection (e.g. transplanted cells), or immune reprogramming (e.g. dendritic cells).
  • the targeting domain may be engineered to target one or more molecules which include but are not limited to immune modulators (e.g. PD-L1, CTLA-4, TIGIT, TIM-3, VISTA, BTLA, LAG3, CD28) and lineage-specific antigens (e.g mesothelin, CD19, HER2).
  • the targeting domain can be made from an antibody, a derivative or any other targeting moiety (e.g.
  • scFv nanobody, single-domain antibody, affitin, affibody, aptamers
  • a natural cellular receptor that is known to interact with the targeted molecule (e.g. PD-1, CD80/CD86, EGF).
  • the chimeric protein of the present invention may be engineered to target one or more molecules involved in immune inhibition, including for example: CSF1 R, CTLA-4, PD-L1 , PD-L2, PD-1 , BTLA, HVEM, TIM3, GAL9, VISTA/VSIG8, KIR, 2B4, TIGIT, CD 160 (also referred to as BY55), CHK 1 and CHK2 kinases, A2aR, CEACAM (e.g., CEACAM-1 , CEACAM-3 and/or CEACAM-5), and various B-7 family ligands (including, but are not limited to, B7-1 , B7-2, B7-DC, B7-H1 , B7-H2, B7-H3, B7-H4, B7-H5, B7-H6 and B7-H7).
  • CSF1 R CSF1 R
  • CTLA-4 CTLA-4
  • the chimeric protein of the present invention may be engineered to target one or more molecules including without limitation, one or more of TIM-3, BTLA, PD-1, CSF1 R, CTLA-4, CD244, CD160, TIGIT, CD172a (SIRP1 a), 2B4, VISTA, VSIG8, CD200 and TMIGD2.
  • the chimeric protein of the present invention may be engineered to target one or more molecules that reside on human leukocytes, myeloid cell, endothelial cells including, without limitation, the extracellular domains of 0X40, SLAMF4, IL-2 R a, 4-1 BB/TNFRSF9, IL-2 R b, ALCAM, BTLA, B7-1, IL-4 R, B7-H3, BLAME/SLAMFS, CEACAM 1 , IL-6 R, IL-7 Ra, IL-10R a, IL-10R b, IL-12 R b 1 , IL-12 R b 2, CD2, IL-13 R a 1, IL-13, CD3, CD4, ILT2/CDS5j, ILT3/CDS5k, ILT4/CDS5d, ILT5/CDS5a, lutegrin a 4/CD49d, CDS, Integrin a E/CD103, CD6, Integrin a M/CD 1 1 b, CDS, Integrin a M
  • leukocytes also known as white blood cells (WBCs) or leucocytes, are the cells of the immune system that are involved in protecting the body against both infectious disease and foreign invaders. All leukocytes cells are produced and derived from multipotent cells in the bone marrow known as hematopoietic stem cells. Leukocytes are found throughout the body, including the blood and lymphatic system.
  • WBCs white blood cells
  • leucocytes are the cells of the immune system that are involved in protecting the body against both infectious disease and foreign invaders. All leukocytes cells are produced and derived from multipotent cells in the bone marrow known as hematopoietic stem cells. Leukocytes are found throughout the body, including the blood and lymphatic system.
  • myeloid cell refers to nucleated hematopoietic cells in the body, consisting of a range of cell types with diverse functions. They include monocytes, macrophages, dendritic cells and granulocytes, and make up a critical arm of the immune system.
  • lymphoid cell refers to cell that play role in the immune system. There are three different lymphocyte lineages: B and T lymphocytes, small in size, and NK lymphocytes, large and granular. Lymphocytes are small white blood cells (leukocytes) found mainly in the lymph nodes and the spleen.
  • fibroblast refers to a type of biological cell that synthesizes the extracellular matrix and collagen,[l] produces the structural framework (stroma) for animal tissues, and plays a critical role in wound healing. [2] Fibroblasts are the most common cells of connective tissue in animals.
  • endothelial cell refers to a permeable barrier for the blood vessel and is involved in the regulation of blood flow. Endothelial cells are pivotal to applications related to wound healing, angiogenesis, inflammatory processes, blood brain barriers, diabetes and other cardiovascular diseases.
  • PD-L1 also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1) refers to a protein that in humans is encoded by the CD274 gene (UniProt number: Q9NZQ7).
  • PD-L1 is a 40kDa type 1 transmembrane protein that has been speculated to play a major role in suppressing the adaptive arm of immune system.
  • CTLA-4 cytotoxic T-lymphocyte-associated protein 4
  • CD 152 cluster of differentiation 152
  • CTLA4 cytotoxic T-lymphocyte-associated protein 4
  • PI 6410 cytotoxic T-lymphocyte-associated protein 4
  • T cell immunoreceptor with Ig and ITIM domains refers to an immune receptor present on some T cells and natural killer cells (NK) (UniProt number: Q495A1).
  • T-cell immunoglobulin and mucin-domain containing-3 refers to a protein that in humans is encoded by the HAVCR2 gene (UniProt number: Q8TDQ0).
  • V-domain immunoglobulin [Ig] -containing suppressor of T- cell activation refers to a type I transmembrane protein that functions as an immune checkpoint and is encoded by the C10orf54 gene (UniProt number: Q9H7M9).
  • B- and T-lymphocyte attenuator refers to a protein that in humans is encoded by the BTLA gene.
  • BTLA has also been designated as CD272 (cluster of differentiation 272) (UniProt number: Q7Z6A9).
  • lymphocyte activation gene 3 refers to a protein which in humans is encoded by the LAG3 gene and is also designated CD223 (cluster of differentiation 223) (Uniprot number: PI 8627).
  • CD28 Cluster of Differentiation 28 refers to a protein expressed on T cells that provide co-stimulatory signals required for T cell activation and survival (Uniprot number: PI 0747).
  • mesothelin also known as MSLN refers to a protein that in humans is encoded by the MSLN gene.
  • Mesothelin is a 40 kDa protein that is expressed in mesothelial cells (Uniprot number: Q13421).
  • B-lymphocyte antigen CD 19 (CD 19), also known as CD 19 molecule (Cluster of Differentiation 19), B-Lymphocyte Surface Antigen B4, T-Cell Surface Antigen Leu- 12 and CVID3 refers to a transmembrane protein that in humans is encoded by the gene CD19. In humans, CD19 is expressed in all B lineage cells. CD19 (Uniprot number: P15391).
  • Receptor tyrosine-protein kinase erbB-2 also known as CD340 (cluster of differentiation 340), proto-oncogene Neu, Erbb2 (rodent), or ERBB2 (human), is a protein that in humans is encoded by the ERBB2 gene.
  • ERBB is abbreviated from erythroblastic oncogene B, a gene isolated from avian genome. It is also frequently called HER2 (from human epidermal growth factor receptor 2) or HER2/neu.HER2”.
  • HER2 refers to a member of the human epidermal growth factor receptor (HER/EGFR/ERBB) family. Amplification or over-expression of this oncogene has been shown to play an imp Uniprot number: P04626).
  • the targeting domain is an antibody.
  • antibody is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
  • the term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMTP ("small modular immunopharmaceutical” scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"
  • Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments.
  • Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001 ; Reiter et al., 1996; and Young et al., 1995 further describe and enable the production of effective antibody fragments.
  • the antibody is a “chimeric” antibody as described in U.S. Pat. No. 4,816,567.
  • the antibody is a humanized antibody, such as described U.S. Pat. Nos. 6,982,321 and 7,087,409.
  • the antibody is a human antibody.
  • a “human antibody” such as described in US 6,075,181 and 6,150,584.
  • the antibody is a single domain antibody such as described in EP 0 368 684, WO 06/030220 and WO 06/003388.
  • the targeting domain is a monoclonal antibody.
  • Monoclonal antibodies can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique, the human B-cell hybridoma technique and the EBV-hybridoma technique.
  • the term "intrabody” generally refer to an intracellular antibody or antibody fragment.
  • Antibodies in particular single chain variable antibody fragments (scFv)
  • scFv single chain variable antibody fragments
  • Such modification may entail for example, the fusion to a stable intracellular protein, such as, e.g., maltose binding protein, or the addition of intracellular trafficking/localization peptide sequences, such as, e.g., the endoplasmic reticulum retention.
  • the intrabody is a single domain antibody.
  • the antibody according to the invention is a single domain antibody.
  • single domain antibody refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb. In some embodiment, the antibody can be any blocking antibody.
  • the targeting domain is a single-domain antibody.
  • single domain antibody has its general meaning in the art and refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such single domain antibody are also called VHH or “nanobody®”.
  • VHH single domain antibody
  • single domain antibody are also called VHH or “nanobody®”.
  • VHH single domain antibody
  • (single) domain antibodies reference is also made to the prior art cited above, as well as to EP 0 368 684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), Holt et al., Trends Biotechnok, 2003, 21(11):484-490; and WO 06/030220, WO 06/003388.
  • the nanobody has a molecular weight approximately one-tenth that of a human IgG molecule, and the protein has a physical diameter of only a few nanometers.
  • One consequence of the small size is the ability of camelid nanobodies to bind to antigenic sites that are functionally invisible to larger antibody proteins, i.e. , camelid nanobodies are useful as reagents to detect antigens that are otherwise cryptic using classical immunological techniques, and as possible therapeutic agents.
  • a nanobody can inhibit as a result of binding to a specific site in a groove or narrow cleft of a target protein, and hence can serve in a capacity that more closely resembles the function of a classical low molecular weight drug than that of a classical antibody.
  • nanobodies being extremely thermostable, stable to extreme pH and to proteolytic digestion, and poorly antigenic. Another consequence is that nanobodies readily move from the circulatory system into tissues, and even cross the blood-brain barrier and can treat disorders that affect nervous tissue. Nanobodies can further facilitated drug transport across the blood brain barrier. See U.S. patent application 20040161738 published August 19, 2004. These features combined with the low antigenicity to humans indicate great therapeutic potential.
  • the amino acid sequence and structure of a single domain antibody can be considered to be comprised of four framework regions or "FRs” which are referred to in the art and herein as “Framework region 1" or “FR1 as “Framework region 2" or “FR2”; as “Framework region 3 " or “FR3”; and as “Framework region 4" or “FR4” respectively; which framework regions are interrupted by three complementary determining regions or “CDRs”, which are referred to in the art as "Complementarity Determining Region for "CDR1”; as “Complementarity Determining Region 2" or “CDR2” and as “Complementarity Determining Region 3" or “CDR3”, respectively.
  • the single domain antibody can be defined as an amino acid sequence with the general structure : FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4 respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3.
  • the amino acid residues of the single domain antibody are numbered according to the general numbering for VH domains given by the International ImMunoGeneTics information system aminoacid numbering (http://imgt.cines.fr/).
  • the antigen binding fragment of the invention is grafted into non-immunoglobulin based antibodies also called antibody mimetics selected from the group consisting of an affibody, an affilin, an affitin, an adnectin, an atrimer, an evasin, a DARPin, an anticalin, an avimer, a fynomer, and a versabody.
  • non-immunoglobulin based antibodies also called antibody mimetics selected from the group consisting of an affibody, an affilin, an affitin, an adnectin, an atrimer, an evasin, a DARPin, an anticalin, an avimer, a fynomer, and a versabody.
  • the chimeric protein of the present invention comprises an extracellular domain of a Type I membrane protein which has immune inhibitory properties.
  • the chimeric protein is engineered to disrupt, block, reduce, and/or inhibit the transmission of an immune inhibitory signal.
  • the present chimeric proteins are capable of, or find use in methods involving, masking an inhibitory ligand on the surface of a tumor cell and replacing that immune inhibitory ligand with an immune stimulatory ligand.
  • the present chimeric proteins in embodiments are capable of, or find use in methods involving, reducing or eliminating an inhibitory immune signal and/or increasing or activating an immune stimulatory signal.
  • a tumor cell bearing an inhibitory signal (and thus evading an immune response) may be substituted for a positive signal binding on a T cell that can then attack a tumor cell.
  • an inhibitory immune signal is masked by the present constructs and a stimulatory immune signal is activated.
  • beneficial properties are enhanced by the single construct approach of the present chimeric proteins.
  • the chimeric protein of the present invention blocks, reduces and/or inhibits PD-1 and PD-L1 or PD-L2 and/or the binding of PD-1 with PD-L1 or PD-L2. In some embodiments, the present chimeric protein blocks, reduces and/or inhibits the activity of CTLA-4 and/or the binding of CTLA-4 with one or more of AP2M 1, CD80, CD86, SHP-2, and PPP2R5A. In some embodiments, the chimeric protein of the present invention increases and/or stimulates GITR and/or the binding of GITR with one or more of GITR ligand.
  • the chimeric protein of the present invention increases and/or stimulates 0X40 and/or the binding of 0X40 with one or more of 0X40 ligand.
  • the chimeric proteins of the present invention are capable of, or find use in methods involving, enhancing, restoring, promoting and/or stimulating immune modulation.
  • the chimeric proteins of the present invention described herein restore, promote and/or stimulate the activity or activation of one or more immune cells against tumor cells including, but not limited to: T cells, cytotoxic T lymphocytes, T helper cells, natural killer (NK) cells, natural killer T (NKT) cells, anti-tumor macrophages (e.g., Ml macrophages), B cells, and dendritic cells.
  • the chimeric proteins of the present invention enhance, restore, promote and/or stimulate the activity and/or activation of T cells, including, by way of a non limiting example, activating and/or stimulating one or more T- cell intrinsic signals, including a pro-survival signal; an autocrine or paracrine growth signal; a p38 MAPK-, ERK-, STAT-, JAK-, ART- or PI3K-mediated signal; an anti-apoptotic signal; and/or a signal promoting and/or necessary for one or more of: proinflammatory cytokine production or T cell migration or T cell tumor infiltration.
  • T- cell intrinsic signals including a pro-survival signal; an autocrine or paracrine growth signal; a p38 MAPK-, ERK-, STAT-, JAK-, ART- or PI3K-mediated signal; an anti-apoptotic signal; and/or a signal promoting and/or necessary for one or more of: proinflammatory cytokine production or T
  • the chimeric proteins of the present invention are capable of, or find use in methods involving, causing an increase of one or more of T cells (including without limitation cytotoxic T lymphocytes, T helper cells, natural killer T (NKT) cells), B cells, natural killer (NIC) cells, natural killer T (NKT) cells, dendritic cells, monocytes, and macrophages (e.g., one or more of Ml and M2) into a tumor or the tumor microenvironment.
  • T cells including without limitation cytotoxic T lymphocytes, T helper cells, natural killer T (NKT) cells), B cells, natural killer (NIC) cells, natural killer T (NKT) cells, dendritic cells, monocytes, and macrophages (e.g., one or more of Ml and M2) into a tumor or the tumor microenvironment.
  • the present chimeric proteins are capable of, or find use in methods involving, inhibiting and/or causing a decrease in recruitment of immunosuppressive cells (e.g., myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), tumor associated neutrophils (TANs), M2 macrophages, and tumor associated macrophages (TAMs)) to the tumor and/or tumor microenvironment (TME).
  • immunosuppressive cells e.g., myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), tumor associated neutrophils (TANs), M2 macrophages, and tumor associated macrophages (TAMs)
  • the present therapies may alter the ratio of Ml versus M2 macrophages in the tumor site and/or TME to favor Ml macrophages.
  • the chimeric proteins of the present invention are capable of, and can be used in methods comprising, inhibiting and/or reducing T cell inactivation and/or immune tolerance to a tumor, comprising administering an effective amount of a chimeric protein described herein to a subject.
  • the chimeric proteins of the present invention are able to increase the serum levels of various cytokines including, but not limited to, one or more of IFNy, TNFa, IL-2, IL- 4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, and IL-22.
  • the present chimeric proteins are capable of enhancing IL-2, IL-4, IL-5, IL-10, IL-13, IL-17A, IL-22, TNFa or IFNy in the serum of a treated subject.
  • administration of the present chimeric protein is capable of enhancing TNFa secretion.
  • administration of the chimeric protein of the present invention is capable of enhancing superantigen mediated TNFa secretion by leukocytes. Detection of such a cytokine response may provide a method to determine the optimal dosing regimen for the indicated chimeric protein.
  • the chimeric proteins of the present invention inhibit, block and/or reduce cell death of an anti-tumor CD8+ and/or CD4+ T cell; or stimulate, induce, and/or increase cell death of a pro-tumor T cell.
  • T cell exhaustion is a state of T cell dysfunction characterized by progressive loss of proliferative and effector functions, culminating in clonal deletion.
  • a pro-tumor T cell refers to a state of T cell dysfunction that arises during many chronic infections and cancer. This dysfunction is defined by poor proliferative and/or effector functions, sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells. Exhaustion prevents optimal control of infection and tumors.
  • an anti-tumor CD8+ and/or CD4+ T cell refers to T cells that can mount an immune response to a tumor.
  • Illustrative pro-tumor T cells include, but are not limited to, Tregs, CD4+ and/or CD8+ T cells expressing one or more checkpoint inhibitory receptors, Th2 cells and Thl7 cells.
  • Checkpoint inhibitory receptors refers to receptors (e.g., CTLA-4, B7-H3, B7-H4, TIM-3) expressed on immune cells that prevent or inhibit uncontrolled immune responses.
  • the present chimeric proteins are capable of, and can be used in methods comprising, increasing a ratio of effector T cells to regulatory T cells.
  • Illustrative effector T cells include ICOS + effector T cells; cytotoxic T cells (e.g., ab TCR, CD3 + , CD8 + , CD45RO + ); CD4 + effector T cells (e.g, ab TCR, CD3 + , CD4 + , CCR7 + , CD62Lhi, IL- 7R/CD127 + ); CD8 + effector T cells (e.g., ab TCR, CD3 + , CD8 + , CCR7 + , CD62Lhi, IL7R/CD127 + ); effector memory T cells (e.g., CD62Llow, CD44 + , TCR, CD3 + , IL 7R/CD127 + , IL-15R + , CCR71ow); central memory T cells (e.g., CCR7 + , CD62L
  • Illustrative regulatory T cells include ICO + regulatory T cells, CD4 + CD25 + FOXP3 + regulatory T cells, CD4 + CD25 + regulatory T cells, CD4 CD25- regulatory T cells, CD4 + CD25high regulatory T cells, TIM-3 + PD-1 + regulatory T cells, lymphocyte activation gene-3 (LAG-3 ) + regulatory T cells, CTLA-4/CD152 + regulatory T cells, neuropilin-1 (Nrp-1 ) + regulatory T cells, CCR4 + CCR + regulatory T cells, CD62L (L-selectin) + regulatory T cells, CD45RBIow regulatory T cells, CD127IOW regulatory T cells, LRRC32/GARP + regulatory T cells, CD39 + regulatory T cells, GITR + regulatory T cells, LAP + regulatory T cells, 1 B1 1 + regulatory T cells, BTLA + regulatory T cells, type 1 regulatory T cells (Trl cells), T helper type 3 (Th3) cells, regulatory cell of natural killer T cell phenotype (NKTregs), CD8 + regulatory
  • the chimeric protein of the invention is composed of a linker.
  • the functional linker (b) connects the domain (a) to the domain (c) with no particular limit of length.
  • the functional linker (b) connects the targeting domain (a) to the signaling domain (c) with no particular limit of length.
  • the functional linker (b) connects the signaling domain (a) to the targeting domain (c) with no particular limit of length.
  • the functional linker (b) connects the domain (c) to the domain (a) with no particular limit of length.
  • the functional linker (b) connects the targeting domain (c) to the signaling domain (a) with no particular limit of length.
  • the functional linker (b) connects the signaling domain (c) to the targeting domain (a) with no particular limit of length.
  • the functional linker (b) connects the targeting/signaling domain (a) to the signaling domain/targeting domain (c) with no particular limit of length.
  • the functional linker (b) connects the targeting/signaling domain (c) to the signaling domain/targeting domain (a) with no particular limit of length.
  • the chimeric protein comprises a linker.
  • the linker may be flexible, including without limitation highly flexible.
  • the linker may be rigid, including without limitation a rigid alpha helix.
  • linker refers to short amino acid sequences created in nature to separate multiple domains in a single protein.
  • the linker may be functional.
  • the linker may function to improve the folding and/or stability, improve the expression, improve the pharmacokinetics, and/or improve the bioactivity of the present chimeric protein.
  • the linker may function to target the chimeric protein to a particular cell type or location.
  • the term “functional linker” refers to a linker having the capacity to activate immune mechanisms by itself.
  • the functional linker of the invention contains peptide sequences of tumor antigens that will be processed and presented on major histocompatibility complex molecules by different cell types, for instance by antigen-presenting cells upon phagocytosis of TAME-IT-targeted cells or directly by targeted cells.
  • the functional linker of the present invention induces an immune response against specific antigens (e.g. tumor- associated antigens, neoantigens, viral antigens).
  • TAA Tumor-Associated Antigens
  • the term “antigen” refers to a molecule or molecular structure, such as may be present on the outside of a pathogen that can be bound by an antigen-specific antibody a B-cell antigen receptor or a T-cell antigen receptor.
  • an antigen-specific antibody a B-cell antigen receptor or a T-cell antigen receptor.
  • the presence of antigens in the body normally triggers an immune response.
  • Antigens are "targeted" by antibodies and T-cell antigen receptors. Each antibody or T-cell antigen receptor is specifically produced by the immune system to match an antigen after cells in the immune system come into contact with it; this allows a precise identification or matching of the antigen and the initiation of an adaptive response.
  • murine antigen refers to a murine molecule or murine molecular structure that can be bound by an antigen-specific antibody, a B-cell antigen receptor or a T-cell antigen receptor.
  • virus antigen refers to a toxin or other substance given off by a virus which causes an immune response in its host.
  • a viral protein is an antigen specified by the viral genome that can be detected by a specific immunological response.
  • Neoantigen refers to mainly tumor-specific antigens generated by mutations in tumor cells, which are only expressed in tumor cells (11). Neoantigens can also be produced by viral infection, alternative splicing and gene rearrangement (12-14). They are ideal targets for T cells to recognize cancer cells and can stimulate strong anti-tumor immune response.
  • the functional linker of the present invention is having a combination of TAA peptides. In some embodiments, the functional linker of the present invention comprises one or multiple TAA peptides. For example, the functional linker comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 TAAs peptides.
  • the functional linker comprises the same TAA peptides.
  • the functional linker comprises the same TAA peptide in at least 1, 2, 3, 4, 5 copies and another TAA peptide in at least 1, 2, 3, 4, 5 copies.
  • the antigens expressed by tumors or pathogenic microorganisms like bacteria and viruses can be inserted as antigenic peptides in the linker (b) of the chimeric protein of the invention. These chimeric proteins can thus be ‘personalized’ for each patient by changing the antigenic peptides of the linker (b) using sequences from the patient’s specific antigens.
  • the linker is selected from a repeats of 4 glycines and 1 serine (G4S sequences, also known as GGGGS), a peptide tags for protein detection with a specific antibody by common laboratory techniques, one or several identified and therapeutically- relevant antigenic peptide sequences that can be presented on class I and class II molecules of the major histocompatibility complex and against which will be directed a specific immune response, for instance Tumor-Associated Antigens (TAA) (e.g. NY-ESO-1, MelanA antigen, MUC-1) or Tolerance Antigens (e.g. MBP, PLP, MOG).
  • TAA Tumor-Associated Antigens
  • MBP MelanA antigen
  • PLP PLP
  • MOG Tolerance Antigens
  • the linker comprising at least one cysteine residue capable of forming a disulfide bond.
  • at least one cysteine residue capable of forming a disulfide bond is, without wishing to be bound by theory, responsible for maintain a proper multimeric state of the chimeric protein and allowing for efficient production.
  • a method of making a stable chimeric protein comprising adjoining a Type I and Type II transmembrane protein extracellular domain with a linker comprising at least one cysteine residue capable of forming a disulfide bond such that the resultant chimeric protein is properly folded and/or forms into a stable multimeric state.
  • the linker may be derived from naturally-occurring multi-domain proteins or are empirical linkers as described, for example, in Chichili et ai, (2013), Protein Sci. 22(2): 153-167, Chen et ai, (2013), Adv Drug Deliv Rev. 65( 10) : 1357-1369, the entire contents of which are hereby incorporated by reference.
  • the linker may be designed using linker designing databases and computer programs such as those described in Chen et al /., (2013), Adv Drug Deliv Rev. 65(10): 1357-1369 and Crasto ef. a/., (2000), Protein Eng. 13(5):309-312, the entire contents of which are hereby incorporated by reference.
  • the linker is a synthetic linker such as (polyethylene glycol) PEG.
  • PEG refers to a polyether compound derived from petroleum. The structure of PEG is commonly expressed as H-(0-CH2-CH2)n-0H. PEG is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (Cl - CIO) alkoxy- or aryloxy -poly ethylene glycol or polyethylene glycol-maleimide.
  • the linker is a polypeptide.
  • the terms “polypeptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well to naturally occurring amino acids polymers and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
  • the linker is comprised a succession of at least 1, 2, 3, 4, 5, 6, 7; 8, 9, 10 Tumor-Associated Antigens (TAA) (e.g. NY-ESO-1, MelanA antigen, MUC-1).
  • TAA Tumor-Associated Antigens
  • the linker is comprised a succession of at least 1, 2, 3, 4, 5, 6, 7; 8, 9, 10 Tolerance Antigens (e.g. MBP, PLP, MOG).
  • the linker is less than about 500 amino acids long, about 450 amino acids long, about 400 amino acids long, about 350 amino acids long, about 300 amino acids long, about 250 amino acids long, about 200 amino acids long, about 150 amino acids long, or about 100 amino acids long.
  • the linker may be less than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 1 1 , about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids long.
  • the linker is flexible. In another embodiment, the linker is rigid.
  • the linker is substantially comprised of glycine and serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97%, or about 98%, or about 99%, or about 100% glycines and serines).
  • the linker is a hinge region of an antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., lgGl , lgG2, lgG3, and lgG4, and IgAl and lgA2)).
  • the hinge region found in IgG, IgA, IgD, and IgE class antibodies, acts as a flexible spacer, allowing the Fab portion to move freely in space.
  • the hinge domains are structurally diverse, varying in both sequence and length among immunoglobulin classes and subclasses. For example, the length and flexibility of the hinge region varies among the IgG subclasses.
  • the hinge region of lgGl encompasses amino acids 216-231 and, because it is freely flexible, the Fab fragments can rotate about their axes of symmetry and move within a sphere centered at the first of two inter- heavy chain disulfide bridges.
  • lgG2 has a shorter hinge than lgGl , with 12 amino acid residues and four disulfide bridges.
  • the hinge region of lgG2 lacks a glycine residue, is relatively short, and contains a rigid poly-proline double helix, stabilized by extra inter-heavy chain disulfide bridges. These properties restrict the flexibility of the lgG2 molecule.
  • lgG3 differs from the other subclasses by its unique extended hinge region (about four times as long as the lgGl hinge), containing 62 amino acids (including 21 prolines and 1 1 cysteines), forming an inflexible poly-proline double helix.
  • the Fab fragments are relatively far away from the Fc fragment, giving the molecule a greater flexibility.
  • the elongated hinge in lgG3 is also responsible for its higher molecular weight compared to the other subclasses.
  • the hinge region of lgG4 is shorter than that of lgGl and its flexibility is intermediate between that of lgGl and lgG2.
  • the linker may be derived from human lgG4 and contain one or more mutations to enhance dimerization (including S228P) or FcRn binding.
  • the immunoglobulin hinge region can be further subdivided functionally into three regions: the upper hinge region, the core region, and the lower hinge region.
  • the upper hinge region includes amino acids from the carboxyl end of Cm to the first residue in the hinge that restricts motion, generally the first cysteine residue that forms an interchain disulfide bond between the two heavy chains.
  • the length of the upper hinge region correlates with the segmental flexibility of the antibody.
  • the core hinge region contains the inter-heavy chain disulfide bridges, and the lower hinge region joins the amino terminal end of the CH2 domain and includes residues in CH2. Id.
  • the core hinge region of wild-type human lgGl contains the sequence Cys-Pro-Pro-Cys which, when dimerized by disulfide bond formation, results in a cyclic octapeptide believed to act as a pivot, thus conferring flexibility.
  • the present linker comprises, one, or two, or three of the upper hinge region, the core region, and the lower hinge region of any antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., lgGl , lgG2, lgG3, and lgG4, and IgAl and lgA2)).
  • the hinge region may also contain one or more glycosylation sites, which include a number of structurally distinct types of sites for carbohydrate attachment.
  • IgAl contains five glycosylation sites within a 17- amino-acid segment of the hinge region, conferring resistance of the hinge region polypeptide to intestinal proteases, considered an advantageous property for a secretory immunoglobulin.
  • the linker of the present invention comprises one or more glycosylation sites.
  • the linker comprises an Fc domain of an antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., lgGl , lgG2, lgG3, and lgG4, and IgAl and lgA2)).
  • the linker comprises a hinge- CH2-CH3 Fc domain derived from a human lgG4 antibody.
  • the linker comprises a hinge-CH2- CH3 Fc domain derived from a human lgGl antibody.
  • the Fc domain exhibits increased affinity for and enhanced binding to the neonatal Fc receptor (FcRn).
  • the Fc domain includes one or more mutations that increases the affinity and enhances binding to FcRn. Without wishing to be bound by theory, it is believed that increased affinity and enhanced binding to FcRn increases the in vivo half-life of the present chimeric proteins.
  • the linker does not comprise an Fc domain of an antibody.
  • personalized therapy relates to information about a person's own genes or proteins to prevent, diagnose, or treat disease.
  • personalized medicine uses specific information about a person's tumor to help make a diagnosis, plan treatment, find out how well treatment is working, or make a prognosis.
  • the linker (b) can be interchanged to adapt to the patient for a personalized therapy perspective.
  • the antigens expressed by tumors or pathogenic microorganisms like bacteria and viruses can be inserted as antigenic peptides in the linker (b) of the chimeric protein of the invention. These chimeric proteins can thus be ‘personalized’ for each patient by changing the antigenic peptides of the linker (b) using sequences from the patient’s specific antigens.
  • the chimeric protein of the invention is composed of a signaling domain (a) and/or a signaling domain (c).
  • the chimeric protein of the invention is composed of a monomeric signaling domain (a) or a dimeric (e.g. homodimeric or heterodimeric) signaling domain (a) or trimeric (e.g. homotrimeric or heterotrimeric) signaling domain (a). In some embodiments, the chimeric protein of the invention is composed of a monomeric signaling domain (c) or a dimeric signaling domain (c) or trimeric signaling domain (c).
  • the term “signaling domain” refers to a domain that binds a surface molecule and initiates an intracellular signal transduction cascade.
  • the ‘signaling domain’ is composed of monomeric, dimeric (e.g. homodimeric or heterodimeric) or trimeric (e.g. homotrimeric or heterotrimeric) proteins of the TNF family and possesses agonistic properties to stimulate the activation of different immune cell types.
  • the signaling domain (c) or the signaling domain (a), in the case of the molecules from the Tumor Necrosis Factor Superfamily (TNFSF), comprises for instance but not limited to CD40L (CD154), 4-1BBL (CD137L) and OX40L (CD252), the active form being a homotrimer of the extracellular domain of the ligand.
  • TNFSF Tumor Necrosis Factor Superfamily
  • TNFSF Tumor Necrosis Factor Superfamily
  • CD40L CD154
  • 4-1BBL CD137L
  • OX40L CD252
  • CD40 Cluster of differentiation 40
  • CD40L CD 154
  • CD137L Cluster of differentiation 137 L
  • 4-1BBL is a member of the tumor necrosis factor (TNF) receptor family and is expressed by activated T cells of both the CD4+ and CD8+ lineages.
  • 4-1 BB Lis also expressed on dendritic cells, B cells, NK cells, neutrophils and macrophages (UniProt number: Q07011).
  • CD137 ligand is mainly expressed on professional antigen-presenting cells (APCs) such as dendritic cells, monocytes/macrophages, and B cells, and its expression is upregulated during activation of these cells.
  • APCs professional antigen-presenting cells
  • its expression has been documented on a variety of hematopoietic cells and non-hematopoietic cells.
  • 4-1 BBL / CD137L is constitutively expressed on many types of cells but its expression levels are low except for a few types of cells.
  • CD137 also known as 4-1 BB and TNFRSF9
  • 4-1 BBL / CD137L is coexpressed with CD137 (also known as 4-1 BB and TNFRSF9) on various types of cells, but expression of CD137 / 4-1 BB potently downregulates that of 4-1 BBL / CD137L by cis-interactions between the two molecules resulting in endocytosis of 4-1 BBL / CD137L (see Byungsuk Kwon et al. Is CD 137 Ligand (CD137L) "Signaling a Fine Tuner of Immune Responses?” Immune Netw. 2015 Jun;15(3):121 -124).
  • TNFRSF4 Tumor necrosis factor receptor superfamily, member 4
  • CD 134 and 0X40 receptor are members of the TNFR- superfamily of receptors which is not constitutively expressed on resting naive T cells, unlike CD28 (UniProt number: P43489).
  • the signaling domain (c) or the signaling domain (a) acts as a specific agonist for receptors expressed on the immune cells of interest (e.g. dendritic cells, T cells including Tregs, macrophages, NK cells, myeloid-derived suppressor cells) depending on the pathological context.
  • the signaling domain (c) or the signaling domain (a) consists in an active form (monomeric or multimeric) of the extracellular domains of immune ligands (e.g. TNFSF family, PD-L1, CTLA-4).
  • DCs dendritic cells
  • antigen-presenting cells also known as accessory cells
  • T cells T cells of the immune system. They act as messengers between the innate and the adaptive immune systems.
  • T cells refers to a type of lymphocyte.
  • T cells are one of the important white blood cells of the immune system and play a central role in the adaptive immune response. T cells can be easily distinguished from other lymphocytes by the presence of a T-cell receptor (TCR) on their cell surface.
  • TCR T-cell receptor
  • T cells are bom from hematopoietic stem cells, found in the bone marrow. Then, developing T cells migrate to the thymus gland to mature. T cells derive their name from this organ where they develop (or mature). After migration to the thymus, the precursor cells mature into several distinct types of T cells. T cell differentiation also continues after they have left the thymus.
  • CD8+ T cells also known as “killer T cells”
  • helper T cells are cytotoxic - this means that they are able to directly kill virus-infected cells, as well as cancer cells.
  • CD8+ T cells are also able to use small signaling proteins, known as cytokines, to recruit other types of cells when mounting an immune response.
  • cytokines small signaling proteins
  • CD4+ helper T cells function by indirectly killing cells identified as foreign: they determine if and how other parts of the immune system respond to a specific, perceived threat.
  • Helper T cells also use cytokine signaling to influence regulatory B cells directly, and other cell populations indirectly.
  • the regulatory T cells (Tregs), formerly known as suppressor T cells refers to a subpopulation of T cells that modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease.
  • Tregs are immunosuppressive and generally suppress or downregulate induction and proliferation of effector T cells.
  • Tregs express the biomarkers CD4, FOXP3, and CD25 and are thought to be derived from the same lineage as naive CD4 cells.
  • NK cells also known as large granular lymphocytes (LGL) refers to a type of cytotoxic lymphocyte critical to the innate immune system that belong to the rapidly expanding family of innate lymphoid cells (ILC) and represent 5-20% of all circulating lymphocytes in humans.
  • the role of NK cells is analogous to that of cytotoxic T cells in the vertebrate adaptive immune response. NK cells provide rapid responses to virus-infected cells, acting at around 3 days after infection, and respond to tumor formation.
  • immune cells detect the major histocompatibility complex (MHC) presented on infected cell surfaces, triggering cytokine release, causing the death of the infected cell by lysis or apoptosis.
  • MHC major histocompatibility complex
  • NK cells are unique, however, as they have the ability to recognize and kill stressed cells in the absence of antibodies and MHC, allowing for a much faster immune reaction. They were named “natural killers” because of the notion that they do not require activation to kill cells that are missing "self markers of MHC class 1. This role is especially important because harmful cells that are missing MHC I markers cannot be detected and destroyed by other immune cells, such as T lymphocyte cells.
  • NK cells can be identified by the presence of CD56 and the absence of CD3 (CD56+, CD3-).
  • NK cells (belonging to the group of innate lymphoid cells) are one of the three kinds of cells differentiated from the common lymphoid progenitor, the other two being B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus, where they then enter into the circulation.
  • MDSC myeloid-derived suppressor cells
  • myeloid lineage a family of cells that originate from bone marrow stem cells.
  • MDSCs strongly expand in pathological situations such as chronic infections and cancer, as a result of an altered haematopoiesis.
  • MDSCs are discriminated from other myeloid cell types in which they possess strong immunosuppressive activities rather than immunostimulatory properties. Similar to other myeloid cells, MDSCs interact with other immune cell types including T cells, dendritic cells, macrophages and natural killer cells to regulate their functions.
  • the chimeric protein of the present invention comprises a trimer of extracellular domains of immune stimulatory signals which is are one or more of 4-1 BBL, OX-40 ligand (OX-40L), LIGHT (CD258), GITR ligand (GITRL), CD70, CD30 ligand, CD40 ligand (CD40L), CD 137 ligand, TRAIL, and TL1A.
  • the chimeric protein of the present invention comprises a combination of three identical immune stimulatory signals, wherein the immune stimulatory signals are chosen from, but are not limited to, 4-1 BBL, OX-40 ligand (OX-40L), LIGHT (CD258), GITR ligand (GITRL), CD70, CD30 ligand, CD40 ligand (CD40L), CD 137 ligand, TRAIL, and TL1A.
  • the immune stimulatory signals are chosen from, but are not limited to, 4-1 BBL, OX-40 ligand (OX-40L), LIGHT (CD258), GITR ligand (GITRL), CD70, CD30 ligand, CD40 ligand (CD40L), CD 137 ligand, TRAIL, and TL1A.
  • the chimeric protein of the present invention comprises a combination of three different immune stimulatory signals, wherein the immune stimulatory signals are chosen from, but are not limited to, 4-1 BBL, OX-40 ligand (OX-40L), LIGHT (CD258), GITR ligand (GITRL), CD70, CD30 ligand, CD40 ligand (CD40L), CD 137 ligand, TRAIL, and TL1A.
  • the immune stimulatory signals are chosen from, but are not limited to, 4-1 BBL, OX-40 ligand (OX-40L), LIGHT (CD258), GITR ligand (GITRL), CD70, CD30 ligand, CD40 ligand (CD40L), CD 137 ligand, TRAIL, and TL1A.
  • the chimeric protein of the present invention comprises a combination of two identical immune stimulatory signals and one different immune stimulatory signals, , wherein the immune stimulatory signals are chosen from, but are not limited to, 4-1 BBL, OX-40 ligand (OX-40L), LIGHT (CD258), GITR ligand (GITRL), CD70, CD30 ligand, CD40 ligand (CD40L), CD 137 ligand, TRAIL, and TL1A.
  • the immune stimulatory signals are chosen from, but are not limited to, 4-1 BBL, OX-40 ligand (OX-40L), LIGHT (CD258), GITR ligand (GITRL), CD70, CD30 ligand, CD40 ligand (CD40L), CD 137 ligand, TRAIL, and TL1A.
  • the chimeric protein of the present invention comprises a trimer of extracellular domains of immune stimulatory signals modified to improve their folding or ligand/receptor interactions.
  • LIGHT also known as tumor necrosis factor superfamily member 14 (TNFSF14), is a secreted protein of the TNF superfamily. It is recognized by herpesvirus entry mediator (HVEM), as well as decoy receptor 3 (UniProt number: 043557).
  • HVEM herpesvirus entry mediator
  • decoy receptor 3 decoy receptor 3
  • TNFSF18 Tumor necrosis factor ligand superfamily member 18
  • GITRL GITR ligand
  • CD70 Cluster of Differentiation 70
  • CD70 refers to a protein that in humans is encoded by CD70 gene.[l] CD70 is a ligand for CD27. The CD70 protein is expressed on highly activated lymphocytes (like in T- and B-cell lymphomas). (UniProt number: P32970).
  • CD30 ligand also known as TNFRSF8, is a cell membrane protein of the tumor necrosis factor receptor family and tumor marker. The protein encoded by this gene is a cytokine that belongs to the tumor necrosis factor (TNF) ligand family. This cytokine is a ligand for receptor TNFRSF18/AITR/GITR.
  • T lymphocyte survival has been shown to modulate T lymphocyte survival in peripheral tissues. This cytokine is also found to be expressed in endothelial cells, and is thought to be important for interaction between T lymphocytes and endothelial cells. (UniProt number: P28908).
  • TNF-related apoptosis-inducing ligand also been designated CD253 (cluster of differentiation 253) and TNFSF10 (tumor necrosis factor (ligand) superfamily, member 10) refers a protein functioning as a ligand that induces the process of cell death called apoptosis.
  • TRAIL is a cytokine that is produced and secreted by most normal tissue cells. It causes apoptosis primarily in tumor cells, by binding to certain death receptors. (UniProt number: P50591).
  • TNFSF15 Tumor necrosis factor ligand superfamily member 15
  • TL1 A Tumor necrosis factor ligand superfamily member 15
  • TNF tumor necrosis factor
  • trimeric protein refers to a macromolecular complex formed by three, usually non-covalently bound, macromolecules like proteins or nucleic acids. A homotrimer would be formed by three identical molecules. A heterotrimer would be formed by three different macromolecules. In particular, the term “trimeric” refers to a repetition (x3) of the sequence of the extracellular domain of each of these molecules and allowing optimal activation of the cells of interest through efficient binding to their trimerized cognate receptors.
  • the signaling domain (c) or the signaling domain (a) is a trimeric protein.
  • the signaling domain (a) or (c) of the invention comprises a trimeric immune stimulatory agent as follows: trimeric 4-1 BBL; trimeric OX-40L; trimeric LIGHT; trimeric GITRL; trimeric CD70; trimeric CD30Ltrimeric CD40L; trimeric HVEM, trimeric GITR; trimeric CD27; trimeric CD28, trimeric CD 137 and trimeric TL1A.
  • the signaling domain of the invention comprises a trimeric 4-1 BBL.
  • the signaling domain of the invention comprises three monomeric 4-1 BBL linked together by a linker, wherein the linker is selected from a repeats of 4 glycines and 1 serine (G4S sequences), a peptide tags for protein detection with a specific antibody by common laboratory techniques, one or several identified and therapeutically- relevant antigenic peptide sequences that can be presented on class I and class II molecules of the major histocompatibility complex and against which will be directed a specific immune response, for instance Tumor-Associated Antigens (TAA) (e.g. NY-ESO-1, MelanA antigen, MUC-1) or Tolerance Antigens (e.g. MBP, PLP, MOG).
  • TAA Tumor-Associated Antigens
  • MBP MelanA antigen
  • PLP e.g. MBP, PLP, MOG
  • MOG Tolerance Antigens
  • the signaling domain of the invention comprises a trimeric immune stimulatory agent with two monomers of 4-1 BBL and one monomeric OX-40L, wherein each monomers of 4-1 BBL are linked together by a linker, wherein the linker is selected from a repeats of 4 glycines and 1 serine (G4S sequences), a peptide tags for protein detection with a specific antibody by common laboratory techniques, one or several identified and therapeutically-relevant antigenic peptide sequences that can be presented on class I and class II molecules of the major histocompatibility complex and against which will be directed a specific immune response, for instance Tumor-Associated Antigens (TAA) (e.g. NY-ESO-1, MelanA antigen, MUC-1) or Tolerance Antigens (e.g. MBP, PLP, MOG).
  • TAA Tumor-Associated Antigens
  • MBP MelanA antigen
  • PLP PLP
  • MOG Tolerance Antigens
  • the signaling domain of the invention comprises a trimeric OX-40L.
  • the signaling domain of the invention comprises three monomeric OX-40L linked together by a linker, wherein the linker is selected from a repeats of 4 glycines and 1 serine (G4S sequences), a peptide tags for protein detection with a specific antibody by common laboratory techniques, one or several identified and therapeutically- relevant antigenic peptide sequences that can be presented on class I and class II molecules of the major histocompatibility complex and against which will be directed a specific immune response, for instance Tumor-Associated Antigens (TAA) (e.g. NY-ESO-1, MelanA antigen, MUC-1) or Tolerance Antigens (e.g. MBP, PLP, MOG).
  • TAA Tumor-Associated Antigens
  • MBP MelanA antigen
  • PLP PLP
  • MOG Tolerance Antigens
  • the signaling domain of the invention comprises a trimeric CD40L.
  • the signaling domain of the invention comprises three monomeric CD40L linked together by a linker, wherein the linker is selected from a repeats of 4 glycines and 1 serine (G4S sequences), a peptide tags for protein detection with a specific antibody by common laboratory techniques, one or several identified and therapeutically- relevant antigenic peptide sequences that can be presented on class I and class II molecules of the major histocompatibility complex and against which will be directed a specific immune response, for instance Tumor-Associated Antigens (TAA) (e.g. NY-ESO-1, MelanA antigen, MUC-1) or Tolerance Antigens (e.g. MBP, PLP, MOG).
  • TAA Tumor-Associated Antigens
  • MBP MelanA antigen
  • PLP PLP
  • MOG Tolerance Antigens
  • trimeric relates to a chemical compound or molecule consisting of three identical simpler molecules or three different simpler molecules, or two identical simpler molecules and one different simpler molecule. In some embodiment, the term “trimeric” includes homotrimeric or heterotrimeric.
  • the signaling domain (c) or the signaling (a) is monomeric.
  • the term “monomeric” relates to a chemical compound or molecule consisting of one simpler molecule.
  • the signaling domain (c) or the signaling (a) is dimeric.
  • the term “dimeric” relates to a chemical compound or molecule consisting of two identical simpler molecule or two different simpler molecule. In some embodiment, the term “dimeric” includes homodimeric or heterodimeric.
  • the present invention relates to a vector for delivery of a heterologous nucleic acid, wherein the nucleic acid encodes the chimeric protein of the present invention.
  • the vectorization of the nucleic acid encoding the chimeric protein or of the chimeric protein of the present invention is used any expression system, and is not limited to replicating and non-replicating viruses (e.g. oncolytic viruses, AAV, lentiviruses), mRNA, plasmids and other gene therapy vectors.
  • replicating and non-replicating viruses e.g. oncolytic viruses, AAV, lentiviruses
  • mRNA e.g. oncolytic viruses, AAV, lentiviruses
  • plasmids e.g., plasmids and other gene therapy vectors.
  • the present invention relates to a vesicular stomatitis virus (VSV) comprising a nucleic acid encoding the chimeric protein having a general structure of N terminus - (a) - (b) - (c) - C terminus wherein (a) is a domain, (b) is a functional linker and (c) is a domain.
  • VSV vesicular stomatitis virus
  • the present invention relates to a vesicular stomatitis virus (VSV) comprising a nucleic acid encoding the chimeric protein having a general structure of N terminus - (c) - (b) - (a) - C terminus wherein (a) is a domain, (b) is a functional linker and (c) is a domain.
  • VSV vesicular stomatitis virus
  • the present invention relates to vesicular stomatitis virus (VSV) comprising a nucleic acid encoding the chimeric protein having a general structure of N terminus - (a) - (b) - (c) - C terminus wherein (a) is a monomeric domain, (b) is a functional linker and (c) is a trimeric domain.
  • VSV vesicular stomatitis virus
  • the present invention relates to vesicular stomatitis virus (VSV) comprising a nucleic acid encoding the chimeric protein having a general structure of N terminus - (c) - (b) - (a) - C terminus wherein (c) is a monomeric domain, (b) is a functional linker and (a) is a trimeric domain.
  • VSV vesicular stomatitis virus
  • the present invention relates to vesicular stomatitis virus (VSV) comprising a nucleic acid encoding the chimeric protein having a general structure of N terminus - (a) - (b) - (c) - C terminus wherein (a) is a monomeric domain, (b) is a functional linker and (c) is a dimeric domain.
  • VSV vesicular stomatitis virus
  • the present invention relates to vesicular stomatitis virus (VSV) comprising a nucleic acid encoding the chimeric protein having a general structure of N terminus - (c) - (b) - (a) - C terminus wherein (c) is a monomeric domain, (b) is a functional linker and (a) is a dimeric domain.
  • VSV vesicular stomatitis virus
  • the present invention relates to a vesicular stomatitis virus (VSV) comprising a chimeric protein having a general structure of N terminus - (a) - (b) - (c) - C terminus wherein (a) is a domain, (b) is a functional linker and (c) is a domain.
  • VSV vesicular stomatitis virus
  • the present invention relates to a vesicular stomatitis virus (VSV) comprising a chimeric protein having a general structure of N terminus - (c) - (b) - (a) - C terminus wherein (a) is a domain, (b) is a functional linker and (c) is a domain.
  • VSV vesicular stomatitis virus
  • the present invention relates to vesicular stomatitis virus (VSV) comprising a chimeric protein having a general structure of N terminus - (a) - (b) - (c) - C terminus wherein (a) is a monomeric domain, (b) is a functional linker and (c) is a trimeric domain.
  • VSV vesicular stomatitis virus
  • the present invention relates to vesicular stomatitis virus (VSV) comprising a chimeric protein having a general structure of N terminus - (c) - (b) - (a) - C terminus wherein (c) is a monomeric domain, (b) is a functional linker and (a) is a trimeric domain.
  • VSV vesicular stomatitis virus
  • the present invention relates to vesicular stomatitis virus (VSV) comprising a chimeric protein having a general structure of N terminus - (a) - (b) - (c) - C terminus wherein (a) is a monomeric domain, (b) is a functional linker and (c) is a dimeric domain.
  • VSV vesicular stomatitis virus
  • the present invention relates to vesicular stomatitis virus (VSV) comprising a chimeric protein having a general structure of N terminus - (c) - (b) - (a) - C terminus wherein (c) is a monomeric domain, (b) is a functional linker and (a) is a dimeric domain.
  • VSV vesicular stomatitis virus
  • the present invention relates to a vaccinia virus (VV) comprising a nucleic acid encoding the chimeric protein having a general structure of N terminus - (a) - (b)
  • the present invention relates to a vaccinia virus (VV) comprising a nucleic acid encoding the chimeric protein having a general structure of N terminus - (c) - (b)
  • the present invention relates to vaccinia virus (VV) comprising a nucleic acid encoding the chimeric protein having a general structure of N terminus - (a) - (b)
  • the present invention relates to vaccinia virus (VV) comprising a nucleic acid encoding the chimeric protein having a general structure of N terminus - (c) - (b)
  • the present invention relates to vaccinia virus (VV) comprising a nucleic acid encoding the chimeric protein having a general structure of N terminus - (a) - (b)
  • the present invention relates to vaccinia virus (VV) comprising a nucleic acid encoding the chimeric protein having a general structure of N terminus - (c) - (b)
  • the present invention relates to a vaccinia virus (VV) comprising a nucleic acid encoding the chimeric protein having a general structure of N terminus - (a) - (b)
  • the present invention relates to a vaccinia virus (VV) comprising a nucleic acid encoding the chimeric protein having a general structure of N terminus - (c) - (b)
  • the present invention relates to vaccinia virus (VV) comprising a nucleic acid encoding the chimeric protein having a general structure of N terminus - (a) - (b)
  • the present invention relates to vaccinia virus (VV) comprising a nucleic acid encoding the chimeric protein having a general structure of N terminus - (c) - (b)
  • the present invention relates to vaccinia virus (VV) comprising a nucleic acid encoding the chimeric protein having a general structure of N terminus - (a) - (b)
  • the present invention relates to vaccinia virus (VV) comprising a nucleic acid encoding the chimeric protein having a general structure of N terminus - (c) - (b)
  • the present invention provides an expression vector, comprising a nucleic acid encoding the chimeric protein described herein.
  • the expression vector comprises DNA or RNA.
  • the expression vector is a mammalian expression vector.
  • Prokaryotic vectors include constructs based on E. coli sequences (see, e.g., Makrides, Microbiol Rev 1996, 60:512-538).
  • Non-limiting examples of regulatory regions that can be used for expression in E. coli include lac, trp, Ipp, phoA, recA, tac, T3, T7 and APL.
  • Non limiting examples of prokaryotic expression vectors may include the Agt vector series such as Agtl l (Huynh et ah, in "DNA Cloning Techniques, Vol. I: A Practical Approach," 1984, (D. Glover, ed.), pp.
  • Prokaryotic host-vector systems cannot perform much of the post- translational processing of mammalian cells, however. Thus, eukaryotic host- vector systems may be particularly useful.
  • a variety of regulatory regions can be used for expression of the chimeric proteins in mammalian host cells. For example, the SV40 early and late promoters, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter can be used.
  • CMV cytomegalovirus
  • RSV-LTR Rous sarcoma virus long terminal repeat
  • Inducible promoters that may be useful in mammalian cells include, without limitation, promoters associated with the metallothionein II gene, mouse mammary tumor virus glucocorticoid responsive long terminal repeats (MMTV- LTR), the b-interferon gene, and the hsp70 gene (see, Williams et al., Cancer Res 1989, 49:2735-42; and Taylor et al., Mol Cell Biol 1990, 10:165-75). Heat shock promoters or stress promoters also may be advantageous for driving expression of the chimeric proteins in recombinant host cells.
  • the expression vectors of the invention comprise a nucleic acid encoding the chimeric proteins (and/or additional agents), or a complement thereof, operably linked to an expression control region, or complement thereof, that is functional in a mammalian cell.
  • the expression control region is capable of driving expression of the operably linked blocking and/or stimulating agent encoding nucleic acid such that the blocking and/or stimulating agent is produced in a human cell transformed with the expression vector.
  • Expression control regions are regulatory polynucleotides (sometimes referred to herein as elements), such as promoters and enhancers, that influence expression of an operably linked nucleic acid.
  • An expression control region of an expression vector of the invention is capable of expressing operably linked encoding nucleic acid in a human cell.
  • the cell is a tumor cell. In another embodiment, the cell is a non-tumor cell.
  • the expression control region confers regulatable expression to an operably linked nucleic acid.
  • a signal (sometimes referred to as a stimulus) can increase or decrease expression of a nucleic acid operably linked to such an expression control region.
  • Such expression control regions that increase expression in response to a signal are often referred to as inducible.
  • Such expression control regions that decrease expression in response to a signal are often referred to as repressible.
  • the amount of increase or decrease conferred by such elements is proportional to the amount of signal present; the greater the amount of signal, the greater the increase or decrease in expression.
  • the present invention contemplates the use of inducible promoters capable of effecting high level of expression transiently in response to a cue.
  • inducible promoters capable of effecting high level of expression transiently in response to a cue.
  • a cell transformed with an expression vector for the chimeric protein (and/or additional agents) comprising such an expression control sequence is induced to transiently produce a high level of the agent by exposing the transformed cell to an appropriate cue.
  • Illustrative inducible expression control regions include those comprising an inducible promoter that is stimulated with a cue such as a small molecule chemical compound. Particular examples can be found, for example, in U.S. Pat. Nos.
  • Expression control regions and locus control regions include full- length promoter sequences, such as native promoter and enhancer elements, as well as subsequences or polynucleotide variants which retain all or part of full-length or non-variant function.
  • promoter sequences such as native promoter and enhancer elements
  • subsequences or polynucleotide variants which retain all or part of full-length or non-variant function.
  • the term "functional" and grammatical variants thereof when used in reference to a nucleic acid sequence, subsequence or fragment, means that the sequence has one or more functions of native nucleic acid sequence (e.g., non-variant or unmodified sequence).
  • the chimeric protein of the invention is or can be secreted.
  • operable linkage refers to a physical juxtaposition of the components so described as to permit them to function in their intended manner.
  • the relationship is such that the control element modulates expression of the nucleic acid.
  • an expression control region that modulates transcription is juxtaposed near the 5' end of the transcribed nucleic acid (i.e., "upstream”).
  • Expression control regions can also be located at the 3' end of the transcribed sequence (i.e., "downstream") or within the transcript (e.g., in an intron).
  • Expression control elements can be located at a distance away from the transcribed sequence (e.g., 100 to 500, 500 to 1000, 2000 to 5000, or more nucleotides from the nucleic acid).
  • a specific example of an expression control element is a promoter, which is usually located 5' of the transcribed sequence.
  • Another example of an expression control element is an enhancer, which can be located 5' or 3' of the transcribed sequence, or within the transcribed sequence.
  • a promoter functional in a human cell is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence into mRNA.
  • a promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and typically a TATA box located 25-30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site.
  • a promoter will also typically contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box.
  • An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation.
  • promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter.
  • transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3' terminus of the mature mRNA is formed by site-specific post-translational cleavage and polyadenylation.
  • transcription terminator and polyadenylation signals include those derived from SV40. Introns may also be included in expression constructs.
  • Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, polymer-based systems, DEAE-dextran, viral transduction, the calcium phosphate precipitation method, etc.
  • liposomes include liposomes; natural polymer-based delivery vehicles, such as chitosan and gelatin; viral vectors are also suitable for in vivo transduction.
  • a targeting agent such as an antibody or ligand specific for a tumor cell surface membrane protein.
  • proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g., capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life.
  • the technique of receptor-mediated endocytosis is described, for example, by Wu et al, J. Biol. Chem. 262, 4429- 4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci.
  • gene delivery agents such as, e.g., integration sequences can also be employed.
  • Numerous integration sequences are known in the art (see, e.g., Nunes-Duby et al., Nucleic Acids Res. 26:391 -406, 1998; Sadwoski, J. Bacterid., 165:341-357, 1986; Bestor, Cell, 122(3):322-325, 2005; Plasterk et al., TIG 15:326-332, 1999; Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). These include recombinases and transposases.
  • Cre Shinkamp and Hamilton, J. Mol. Biol., 150:467-486, 1981
  • lambda Nash, Nature, 247, 543-545, 1974
  • Flp Broach, et ak, Cell, 29:227-234, 1982
  • R Matsuzaki, et al., J. Bacteriology, 172:610-618, 1990
  • cpC31 see, e.g., Groth et al, J. Mol. Biol.
  • transposases of the mariner family (Plasterk et al., supra), and components for integrating viruses such as AAV, retroviruses, lentivirus having components that provide for virus integration such as the LTR sequences of retroviruses or lentivirus and the ITR sequences of AAV (Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003).
  • viruses such as AAV, retroviruses, lentivirus having components that provide for virus integration such as the LTR sequences of retroviruses or lentivirus and the ITR sequences of AAV (Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003).
  • direct and targeted genetic integration strategies may be used to insert nucleic acid sequences encoding the chimeric fusion proteins including CRISPR/CAS9, zinc finger, TALEN, and meganuclease gene-editing technologies.
  • the invention provides expression vectors for the expression of the chimeric proteins (and/or additional agents) that are viral vectors.
  • viral vectors useful for gene therapy are known (see, e.g., Lundstrom, Trends Biotechnok, 21 : 1 17, 122, 2003.
  • Illustrative viral vectors include those selected from vesicular stomatitis virus (VSV), lentivirus, (LV), retroviruses (RV), adenoviruses (AV), adeno-associated viruses (AAV) or a virus from the same families, measles virus, herpesvirus, vaccinia virus (VV), myxoma virus, reovirus, parvovirus, mumps virus and a viruses though other viral vectors may also be used.
  • VSV vesicular stomatitis virus
  • LV lentivirus,
  • RV retroviruses
  • AV adenoviruses
  • AAV adeno-associated viruses
  • virus from the same families measles virus, herpesvirus, vaccinia virus (VV), myxoma virus, reovirus, parvovirus, mumps virus and a viruses though other viral vectors may also be used.
  • VV vesicular sto
  • viruses include Sindbis virus, Venezuelan equine encephalitis (VEE) virus, and Semliki Forest virus (SFV), HSV, enterovirus, paramyxovirus, poxvirus, rhabdovirus.
  • VEE Venezuelan equine encephalitis
  • SFV Semliki Forest virus
  • HSV enterovirus
  • paramyxovirus paramyxovirus
  • poxvirus poxvirus
  • rhabdovirus rhabdovirus.
  • viral vectors that integrate into the host genome are suitable, such as retroviruses, AAV, lentivirus.
  • the invention provides methods of transducing a human cell in vivo, comprising contacting a solid tumor in vivo with a viral vector of the invention.
  • the viral vector of the invention is a vesicular stomatitis virus (VSV).
  • VSV vesicular stomatitis virus
  • the viral vector of the invention is a recombinant vaccinia virus (VV).
  • the present invention provides a host cell, comprising the expression vector comprising the chimeric protein described herein.
  • Expression vectors can be introduced into host cells for producing the present chimeric proteins.
  • Cells may be cultured in vitro or genetically engineered, for example.
  • Useful mammalian host cells include, without limitation, cells derived from humans, monkeys, and rodents (see, for example, Kriegler in "Gene Transfer and Expression: A Laboratory Manual," 1990, New York, Freeman & Co.).
  • monkey kidney cell lines transformed by SV40 e.g., COS-7, ATCC CRL 1651
  • human embryonic kidney lines e.g., 293, 293-EBNA, or 293 cells subcloned for growth in suspension culture, Graham et al., J Gen Virol 1977, 36:59
  • baby hamster kidney cells e.g., BHK, ATCC CCL 10
  • Chinese hamster ovary-cells- DHFR e.g., CHO, Urlaub and Chasin, Proc Natl Acad Sci USA 1980, 77:4216
  • DG44 CHO cells CHO-K1 cells, mouse Sertoli cells (Mather, Biol Reprod 1980, 23:243-251)
  • mouse fibroblast cells e.g., NIH-3T3
  • monkey kidney cells e.g., CV1 ATCC CCL 70
  • African green monkey kidney cells e.g., VERO-76, ATCC CRL-1587
  • human cervical carcinoma cells e.g
  • Illustrative cancer cell types for expressing the chimeric proteins described herein include mouse fibroblast cell line, NIH3T3, mouse Lewis lung carcinoma cell line, LLC, mouse mastocytoma cell line, P815, mouse lymphoma cell line, EL4 and its ovalbumin transfectant, E.G7, mouse melanoma cell line, B16F10, mouse fibrosarcoma cell line, MC57, and human small cell lung carcinoma cell lines, SCLC#2 and SCLC#7.
  • Gene delivery viral vectors useful in the practice of the present invention can be constructed utilizing methodologies well known in the art of molecular biology.
  • viral vectors carrying transgenes are assembled from polynucleotides encoding the transgene, suitable regulatory elements and elements necessary for production of viral proteins which mediate cell transduction.
  • Host cells can be obtained from normal or affected subjects, including healthy humans, cancer patients, and patients with an infectious disease, private laboratory deposits, public culture collections such as the American Type Culture Collection, or from commercial suppliers.
  • Cells that can be used for production of the present chimeric proteins in vitro, ex vivo, and/or in vivo include, without limitation, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells (e.g., as obtained from bone marrow), umbilical cord blood, peripheral blood, fetal liver, etc.
  • the choice of cell type depends on the type of tumor or infectious disease being treated or prevented, and can be determined by one of skill in the art.
  • the present invention relates to a method of treatment or prevention of cancers and/or tumors.
  • the treatment of cancer may involve modulating the immune system with the present chimeric proteins to favor immune stimulation over immune inhibition.
  • the present invention relates to a chimeric protein for use in a method of treatment or prevention in a patient suffering from cancer.
  • the present invention relates to a method of treatment of a subject suffering from cancer comprising administered a therapeutically effective amount of a chimeric protein comprising the structure: N terminus - (a) - (b) - (c) - C terminus wherein (a) is a domain, (b) is a functional linker and (c) is a domain.
  • the present invention relates to a method of treatment of a subject suffering from cancer comprising administered a therapeutically effective amount of a chimeric protein comprising the structure: N terminus - (c) - (b) - (a) - C terminus wherein (a) is a domain, (b) is a functional linker and (c) is a domain.
  • the present invention relates to a method of treatment of a subject suffering from cancer comprising administered a therapeutically effective amount of a chimeric protein comprising the structure: N terminus - (a) - (b) - (c) - C terminus wherein (a) is a signaling and/or targeting domain, (b) is a functional linker and (c) is a signaling and/or targeting domain.
  • the present invention relates to a method of treatment of a subject suffering from cancer comprising administered a therapeutically effective amount of a chimeric protein comprising the structure: N terminus - (c) - (b) - (a) - C terminus wherein (a) is a signaling and/or targeting domain, (b) is a functional linker and (c) is a signaling and/or targeting domain.
  • the present invention relates to a method of treatment of a subject suffering from cancer comprising administered a therapeutically effective amount of a chimeric protein comprising the structure: N terminus - (a) - (b) - (c) - C terminus wherein (a) is a monomeric domain, (b) is a functional linker and (c) is a trimeric domain.
  • the present invention relates to a method of treatment of a subject suffering from cancer comprising administered a therapeutically effective amount of a chimeric protein comprising the structure: N terminus - (c) - (b) - (a) - C terminus wherein (c) is a monomeric domain, (b) is a functional linker and (a) is a trimeric domain.
  • the present invention relates to a method of treatment of a subject suffering from cancer comprising administered a therapeutically effective amount of a chimeric protein comprising the structure: N terminus - (a) - (b) - (c) - C terminus wherein (a) is a monomeric domain, (b) is a functional linker and (c) is a dimeric domain.
  • the present invention relates to a method of treatment of a subject suffering from cancer comprising administered a therapeutically effective amount of a chimeric protein comprising the structure: N terminus - (c) - (b) - (a) - C terminus wherein (c) is a monomeric domain, (b) is a functional linker and (a) is a dimeric domain.
  • the term “subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate.
  • the subject according to the invention is a human. More particularly, the subject according to the invention has or is susceptible to have cancer.
  • the term “subject” encompasses “patient”.
  • the term “biological sample” refers to any sample obtained from a subject, such as a serum sample, a plasma sample, a urine sample, a blood sample, a lymph sample, or a tissue biopsy.
  • the biological sample is a tissue biopsy.
  • tissue when used in reference to a part of a body or of an organ, generally refers to an aggregation or collection of morphologically similar cells and associated accessory and support cells and intercellular matter, including extracellular matrix material, vascular supply, and fluids, acting together to perform specific functions in the body.
  • tissue There are generally four basic types of tissue in animals and humans including muscle, nerve, epithelial, and connective tissues.
  • the tissue sample when the subject suffers from a cancer, is a tumor tissue sample.
  • tumor tissue sample means any tissue tumor sample derived from the subject. Said tissue sample is obtained for the purpose of the in vitro evaluation.
  • the tumor sample may result from the tumor resected from the subject.
  • the tumor sample may result from a biopsy performed in the primary tumor of the subject or performed in metastatic sample distant from the primary tumor of the subject.
  • the tumor tissue sample encompasses a global primary tumor (as a whole), a tissue sample from the center of the tumor, a tumor tissue sample collected prior surgery (for follow-up of subjects after treatment for example), and a distant metastasis.
  • the tumor tissue sample can, of course, be subjected to a variety of well-known post-collection preparative and storage techniques (e.g., fixation, storage, freezing, etc.).
  • the sample can be fresh, frozen, fixed (e.g., formalin fixed), or embedded (e.g., paraffin embedded.
  • cancer has its general meaning in the art and refers to a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body.
  • the term “cancer” further encompasses both primary and metastatic cancers.
  • Examples of cancers that may treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acid
  • treating refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).
  • administering refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., an inhibitor of caspase 8 alone or in a combination with a classical treatment) into the subject, such as by, intravenous, intramuscular, enteral, subcutaneous, parenteral, systemic, local, spinal, nasal, topical or epidermal administration (e.g., by injection or infusion).
  • a disease, or a symptom thereof is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof.
  • administration of the substance typically occurs before the onset of the disease or symptoms thereof.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.
  • a therapeutically effective amount of drug may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of drug to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the chimeric protein or chimeric protein portion are outweighed by the therapeutically beneficial effects.
  • the efficient dosages and dosage regimens for drug depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • a suitable dose of a composition of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen.
  • Such an effective dose will generally depend upon the factors described above.
  • a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease.
  • One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.
  • An exemplary, non-limiting range for a therapeutically effective amount of drug is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg/kg, about 5 mg/kg or about 8 mg/kg.
  • An exemplary, non-limiting range for a therapeutically effective amount of an chimeric protein of the present invention is 0.02-100 mg/kg, such as about 0.02- 30 mg/kg, such as about 0.05-10 mg/kg or 0.1-3 mg/kg, for example about 0.5-2 mg/kg. Administration may e.g.
  • treatment may be intravenous, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target.
  • Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time.
  • treatment according to the present invention may be provided as a daily dosage of the agent of the present invention in an amount of about 0.1-100 mg/kg, such as 0.2, 0.5, 0.9,
  • the protein or the gene coding for the chimeric protein of the invention as described above is administered to the subject in the form of a pharmaceutical composition which comprises a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene- block polymers, polyethylene glycol and wool fat.
  • compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, intrapleurally, intraperitoneally or via an implanted reservoir.
  • the used herein includes subcutaneous, intravenous, intramuscular, intra- articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
  • Sterile injectable forms of the compositions of this invention may be aqueous or an oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3- butanediol.
  • a non-toxic parenterally acceptable diluent or solvent for example as a solution in 1,3- butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono-or diglycerides.
  • Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically- acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include, e.g., lactose.
  • the active ingredient is combined with emulsifying and suspending agents.
  • certain sweetening, flavoring or coloring agents may also be added.
  • the compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug.
  • suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug.
  • Such materials include cocoa butter, beeswax and polyethylene glycols.
  • compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.
  • the compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers.
  • Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.
  • compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers.
  • suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol and water.
  • Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Patches may also be used.
  • the compositions of this invention may also be administered by nasal aerosol or inhalation.
  • compositions are prepared according to techniques well- known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
  • an chimeric protein present in a pharmaceutical composition of this invention can be supplied at a concentration of 10 mg/mL in either 100 mg (10 mL) or 500 mg (50 mL) single use vials.
  • the product is formulated for IV administration in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mL polysorbate 80, and Sterile Water for Injection. The pH is adjusted to 6.5.
  • An exemplary suitable dosage range for an chimeric protein in a pharmaceutical composition of this invention may between about 1 mg/m2 and 500 mg/m2.
  • schedules are exemplary and that an optimal schedule and regimen can be adapted taking into account the affinity and tolerability of the particular antibody in the pharmaceutical composition that must be determined in clinical trials.
  • a pharmaceutical composition of the invention for injection e.g., intramuscular, i.v.
  • the term “combination” is intended to refer to all forms of administration that provide a first drug together with a further (second, third%) drug.
  • the drugs may be administered simultaneous, separate or sequential and in any order.
  • the drug is administered to the subject using any suitable method that enables the drug to reach the lungs.
  • the drug administered to the subject systemically (i.e. via systemic administration).
  • the drug is administered to the subject such that it enters the circulatory system and is distributed throughout the body.
  • the drug is administered to the subject by local administration, for example by local administration to the lungs.
  • the term “combination” is intended to refer to all forms of administration that provide a first drug together with a further (second, third%) drug.
  • the present chimeric protein may be administered simultaneously, separately or sequentially and in any order.
  • the present chimeric protein is administered to the subject using any suitable method that enables the drug to reach the kidney.
  • the present chimeric protein is administered to the subject systemically (i.e. via systemic administration).
  • the drug is administered to the subject such that it enters the circulatory system and is distributed throughout the body.
  • the terms “combined treatment”, “combined therapy” or “therapy combination” refer to a treatment that uses more than one medication.
  • the combined therapy may be dual therapy or bi-therapy.
  • the present invention chemical compounds that are effective in inhibiting tumor growth.
  • chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaorarnide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophyc
  • calicheamicin especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Inti. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolin
  • paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisp latin and carbop latin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • antihormonal agents that act to regulate or inhibit honnone action on tumors
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • immunotherapeutic refers to any type of compound which can be used in immunotherapy. Immunotherapy as used herein is the treatment of disease by inducing, enhancing, or suppressing an immune response. An “anti cancer immunotherapy”, as used herein, stimulates the immune system to reject and destroy tumors. As used herein the term “anti-cancer immunotherapeutic” as used herein thus includes such compounds as e.g. Tumor-Specific Antigens (TSA), Tumor-Associated Antigens (TAA), immune adjuvants, immune modulators, antibodies, modified immune cells, cytokines, immune checkpoint blockade molecules, viruses.
  • TSA Tumor-Specific Antigens
  • TAA Tumor-Associated Antigens
  • immune modulators antibodies, modified immune cells, cytokines, immune checkpoint blockade molecules, viruses.
  • immune modulator is a component that modulates the immune responses to an antigen towards the desired immune responses.
  • the present invention includes additional agent which can be one or more immune-modulating agents.
  • additional agent which can be one or more immune-modulating agents.
  • immunotherapeutic agent refers to a compound, composition or treatment that indirectly or directly enhances, stimulates or increases the body's immune response against cancer cells and/or that decreases the side effects of other anticancer therapies. Immunotherapy is thus a therapy that directly or indirectly stimulates or enhances the immune system's responses to cancer cells and/or lessens the side effects that may have been caused by other anti-cancer agents. Immunotherapy is also referred to in the art as immunologic therapy, biological therapy biological response modifier therapy and biotherapy.
  • immunotherapeutic agents examples include, but are not limited to, immune checkpoint inhibitor, cytokines, cancer vaccines, monoclonal antibodies and non-cytokine adjuvants.
  • the immunotherapeutic treatment may consist of administering the subject with an amount of immune cells (T cells, NK, cells, dendritic cells, B cells... ).
  • Immunotherapeutic agents can be non-specific, i.e. boost the immune system generally so that the human body becomes more effective in fighting the growth and/or spread of cancer cells, or they can be specific, i.e. targeted to the cancer cells themselves immunotherapy regimens may combine the use of non-specific and specific immunotherapeutic agents.
  • Non-specific immunotherapeutic agents are substances that stimulate or indirectly improve the immune system.
  • Non-specific immunotherapeutic agents have been used alone as a main therapy for the treatment of cancer, as well as in addition to a main therapy, in which case the non-specific immunotherapeutic agent functions as an adjuvant to enhance the effectiveness of other therapies (e.g. cancer vaccines).
  • Non-specific immunotherapeutic agents can also function in this latter context to reduce the side effects of other therapies, for example, bone marrow suppression induced by certain chemotherapeutic agents.
  • Non-specific immunotherapeutic agents can act on key immune system cells and cause secondary responses, such as increased production of cytokines and immunoglobulins. Alternatively, the agents can themselves comprise cytokines.
  • Non-specific immunotherapeutic agents are generally classified as cytokines or non-cytokine adjuvants.
  • cytokines have found application in the treatment of cancer either as general non-specific immunotherapies designed to boost the immune system, or as adjuvants provided with other therapies. Suitable cytokines include, but are not limited to, interferons, interleukins and colony-stimulating factors. Interferons (IFNs) contemplated by the present invention include the common types of IFNs, IFN-alpha (IFN-a), and IFN-beta (IFN-b). IFNs can act directly on cancer cells, for example, by slowing their growth, promoting their development into cells with more normal behaviour and/or increasing their production of antigens thus making the cancer cells easier for the immune system to recognise and destroy.
  • IFNs Interferons
  • IFN-a IFN-alpha
  • IFN-b IFN-beta
  • IFNs can also act indirectly on cancer cells, for example, by slowing down angiogenesis, boosting the immune system and/or stimulating natural killer (NK) cells, T cells and macrophages.
  • Recombinant IFN-alpha is available commercially as Roferon (Roche Pharmaceuticals) and Intron A (Schering Corporation).
  • Interleukins contemplated by the present invention include IL-2, IL-4, IL-11 and IL-12. Examples of commercially available recombinant interleukins include Proleukin® (IL-2; Chiron Corporation) and Neumega® (IL-12; Wyeth Pharmaceuticals). Zymogenetics, Inc.
  • CSFs Colony-stimulating factors
  • Treatment with one or more growth factors can help to stimulate the generation of new blood cells in subjects undergoing traditional chemotherapy. Accordingly, treatment with CSFs can be helpful in decreasing the side effects associated with chemotherapy and can allow for higher doses of chemotherapeutic agents to be used.
  • immunotherapeutic agents can be active, i.e. stimulate the body's own immune response, or they can be passive, i.e. comprise immune system components that were generated external to the body.
  • Passive specific immunotherapy typically involves the use of one or more monoclonal antibodies that are specific for a particular antigen found on the surface of a cancer cell or that are specific for a particular cell growth factor.
  • Monoclonal antibodies may be used in the treatment of cancer in a number of ways, for example, to enhance a subject's immune response to a specific type of cancer, to interfere with the growth of cancer cells by targeting specific cell growth factors, such as those involved in angiogenesis, or by enhancing the delivery of other anticancer agents to cancer cells when linked or conjugated to agents such as chemotherapeutic agents, radioactive particles or toxins.
  • Monoclonal antibodies currently used as cancer immunotherapeutic agents that are suitable for inclusion in the combinations of the present invention include, but are not limited to, rituximab (Rituxan®), trastuzumab (Herceptin®), ibritumomab tiuxetan (Zevalin®), tositumomab (Bexxar®), cetuximab (C-225, Erbitux®), bevacizumab (Avastin®), gemtuzumab ozogamicin (Mylotarg®), alemtuzumab (Campath®), and BL22.
  • Other examples include anti-CTLA4 antibodies (e.g.
  • Ipilimumab anti-PD 1 antibodies, anti-PDLl antibodies, anti-PLD2 antibodies (selected by way of non-limiting example, one or more of nivolumab (ONO- 4538/BMS-936558, MDX1 106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, Merck), MK- 3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), atezolizumab (TECENTRIQ, GENENTECH), MPDL3280A (ROCHE)), anti-TIMP3 antibodies, anti-LAG3 antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies or anti-B7H6 antibodies.
  • nivolumab ONO- 4538/BMS-936558, MDX1 106, OPDIVO, BRISTOL MYERS SQUIBB
  • pembrolizumab KYTRUDA, Merck
  • MK- 3475
  • antibodies include B cell depleting antibodies.
  • Typical B cell depleting antibodies include but are not limited to anti-CD20 monoclonal antibodies [e.g. Rituximab (Roche), Ibritumomab tiuxetan (Bayer Schering), Tositumomab (GlaxoSmithKline), AME-133v (Applied Molecular Evolution), Ocrelizumab (Roche), Ofatumumab (HuMax-CD20, Gemnab), TRU-015 (Trubion) and IMMU-106 (Immunomedics)], an anti-CD22 antibody [e.g.
  • an agent that increases and/or stimulates CD137 (4-1 BB) and/or the binding of CD137 (4-1 BB) with one or more of 4-1 BB ligand by way of non-limiting example, urelumab (BMS-663513 and anti -4- 1 BB antibody), and an agent that blocks, reduces and/or inhibits the activity of CTLA-4 and/or the binding of CTLA- 4 with one or more of AP2M 1 , CD80, CD86, SHP-2, and PPP2R5A and/or the binding of 0X40 with OX40L (by way of non-limiting example GBR 830 (GLENMARK), MEDI6469 (MEDIMMUNE).
  • GBR 830 GLENMARK
  • MEDI6469 MEDIMMUNE
  • the immunotherapeutic treatment may consist of allografting, in particular, allograft with hematopoietic stem cell HSC.
  • the immunotherapeutic treatment may also consist in an adoptive immunotherapy as described by Nicholas P. Restifo, Mark E. Dudley and Steven A. Rosenberg “Adoptive immunotherapy for cancer: harnessing the T cell response, Nature Reviews Immunology, Volume 12, April 2012).
  • adoptive immunotherapy the subject’s circulating lymphocytes, NK cells, are isolated amplified in vitro and readministered to the subject.
  • the activated lymphocytes or NK cells are most particularly be the subject’s own cells that were earlier isolated from a blood or tumor sample and activated (or “expanded”) in vitro.
  • the chimeric proteins are used to treat, control or prevent one or more inflammatory diseases or conditions.
  • inflammatory diseases include acne vulgaris, acute inflammation, allergic rhinitis, asthma, atherosclerosis, atopic dermatitis, autoimmune disease, autoinflammatory diseases, autosomal recessive spastic ataxia, bronchiectasis, celiac disease, chronic cholecystitis, chronic inflammation, chronic prostatitis, colitis, diverticulitis, familial eosinophilia (fe), glomerulonephritis, glycerol kinase deficiency, hidradenitis suppurativa, hypersensitivities, inflammation, inflammatory bowel diseases, inflammatory pelvic disease, interstitial cystitis, laryngeal inflammatory disease, Leigh syndrome, lichen planus, mast cell activation syndrome, mastocytosis, ocular inflammatory disease, otitis, pain, pelvic
  • the inflammatory disease is an autoimmune disease or condition, such as multiple sclerosis (e.g. autoimmune encephalomyelitis (EAE)), diabetes mellitus, lupus, celiac disease, Crohn's disease, ulcerative colitis, Guillain-Barre syndrome, scleroderms, Goodpasture's syndrome, Wegener's granulomatosis, autoimmune epilepsy, Rasmussen's encephalitis, Primary biliary sclerosis, Sclerosing cholangitis, Autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis, Fibromyalgia, Menier's syndrome; transplantation rejection (e.g., prevention of allograft rejection) pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus
  • the chimeric proteins of the invention when used to treat, control or prevent transplantation rejection, has a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (a) is a signaling and/or targeting domain, (b) is a linker (b) is a functional linker and (c) is a monomeric signaling and/or targeting domain or has a general structure of: N terminus - (c) - (b) - (a) - C terminus, wherein: (c) is a signaling and/or targeting domain, (b) is a linker (b) is a functional linker and (a) is a monomeric signaling and/or targeting domain.
  • the present chimeric agents are used to eliminate intracellular pathogens. In some aspects, the present chimeric agents are used to treat one or more infections.
  • the present chimeric proteins are used in methods of treating viral infections (including, for example, HIV and HCV), parasitic infections (including, for example, malaria), and bacterial infections.
  • the infections induce immunosuppression.
  • HIV infections often result in immunosuppression in the infected subjects.
  • the treatment of such infections may involve, in some embodiments, modulating the immune system with the present chimeric proteins to favor immune stimulation over immune inhibition.
  • the present invention provides methods for treating infections that induce immunoactivation.
  • intestinal helminth infections have been associated with chronic immune activation.
  • the treatment of such infections may involve modulating the immune system with the present chimeric proteins to favor immune inhibition over immune stimulation.
  • administration simultaneously refers to administration of 2 active ingredients by the same route and at the same time or at substantially the same time.
  • administration separately refers to an administration of 2 active ingredients at the same time or at substantially the same time by different routes.
  • administration sequentially refers to an administration of 2 active ingredients at different times, the administration route being identical or different.
  • the chimeric protein of the invention may target a cell (e.g., cancer cell or immune cell) that expresses PD-L1 and/or PD-L2.
  • the chimeric protein may target a cell (e.g., cancer cell or immune cell) that expresses OX-40.
  • the chimeric protein may target a cell (e.g., cancer cell or immune cell) that expresses GITR. In an illustrative embodiment, the chimeric protein may target a cell (e.g., cancer cell or immune cell) that expresses 4-1 BB. In some embodiment, the chimeric protein may target a cell (e.g., cancer cell or immune cell) that expresses CD40. In an illustrative embodiment, the chimeric protein may target a cell (e.g., cancer cell or immune cell) that expresses VISTA. In an illustrative embodiment, the chimeric protein may target a cell (e.g., cancer cell or immune cell) that expresses CSF1. In some embodiment, the chimeric protein may target a cell (e.g., cancer cell or immune cell) that expresses IL-34.
  • the chimeric protein may target a cell (e.g., cancer cell or immune cell) that expresses CD47.
  • the chimeric protein may target a cell (e.g., cancer cell, stromal cell or immune cell) that expresses galectin-9 and/or phosphatidyserine.
  • the present methods provide treatment with the chimeric protein in a patient who is refractory to an additional agent, such "additional agents" being described elsewhere herein, inclusive, without limitation, of the various chemotherapeutic agents described herein.
  • the chimeric proteins of the invention when used to treat, control or prevent cancer or use in immunotherapy, has a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (a) is a signaling and/or targeting domain, (b) is a linker (b) is a functional linker and (c) is a dimeric or trimeric signaling and/or targeting domain or has a general structure of: N terminus - (c) - (b) - (a) - C terminus, wherein: (c) is a signaling and/or targeting domain, (b) is a linker (b) is a functional linker and (a) is a dimeric or trimeric signaling and/or targeting domain.
  • FIGURES are a diagrammatic representation of FIGURES.
  • TAME-IT molecules are chimeric proteins composed of three different domains with distinct immunostimulating activities.
  • the ‘targeting domain’ recognizes a marker on the surface of either tumor cells or immune cells; this anchors and displays the TAME-IT molecule on the cell surface and may signal or block signaling inside the targeted cells.
  • the ‘functional linker’ contains peptide sequences of tumor or viral antigens that will be processed and presented on major histocompatibility complex molecules by different cell types, including by antigen-presenting cells upon phagocytosis of TAME-IT -targeted cells.
  • the ‘signaling domain’ is composed of monomeric, dimeric or trimeric proteins of the TNF family and possesses agonistic properties to stimulate the activation of different immune cell types.
  • TAME-IT proteins display agonistic ligands on the surface of targeted cells.
  • PD-L1 + cells were incubated with the KN035-TAA-th4-lBBL molecule, KN035 being an anti-PD-Ll nanobody and th4-lBBL a trimeric human 4-1BBL domain, washed and then stained with a fluorochrome-conjugated anti-4-lBBL antibody.
  • the TAME-IT molecule KN035-th4-lBBL is able to recognize surface PD-L1 and renders PD-L1 + cells positive for 4- 1BBL (A).
  • CTLA-4 + / CD40L cells were incubated (dark grey) or not (light grey) with an [Anti-CTLA-4]-[thCD40L] TAME- IT molecule, washed and then stained with a fluorochrome-conjugated anti-CD40L antibody.
  • E The TAME-IT molecule renders CTLA-4 + / CD40L cells positive for CD40L due to the fixation on their surface.
  • TAME-IT molecules activate human dendritic cells.
  • A-C Human monocyte-derived dendritic cells were incubated with the supernatants from either KN035-th4- lBBL-producing cells (dark grey) or control cells (light grey) for 48h. Dendritic cells were then analyzed for the expression of CD80, CD83 and CD86 maturation markers by flow cytometry. This shows that the TAME-IT proteins induce the maturation of antigen-presenting cells.
  • FIG. 4 Cells infected by engineered oncolytic viruses produce TAME-IT molecules.
  • HEK cells were infected by with a VSV coding for the Al-thCD40L molecule.
  • the culture supernatant (SN) was collected and concentrated by centrifugation (CSN).
  • CSN centrifugation
  • the Al- thCD40L was then detected by western-blot.
  • mRuby2 a VSV coding for a non- relevant protein
  • Antigens encoded in the ‘functional linker’ are efficiently presented to CD4 T cells.
  • Human tumor cells were infected with a VSV coding for different TAME-IT molecules containing or not a ‘functional linker’ that contains antigenic peptides. These infected cells were then co-cultured or not with human monocyte-derived dendritic cells before being co-cultured for 6 hours with a human, HLA-DP4-restricted, CD4 T cell clone specific for the tumor antigen NY-ESO-1 which peptidic sequence is found in the ‘functional linker’.
  • T cell activation was evaluated by intracellular staining of gamma-interferon and was only observed against HLA-DP4 + dendritic cells (expressing MHC class II molecules) that previously internalized tumor cells infected with the VSV coding for TAME-IT molecules containing a ‘functional linker’. This shows that the antigenic peptides from the TAME-IT molecules are efficiently processed by human antigen-presenting cells and presented to tumor-associated antigen-specific human CD4 T cells.
  • Antigens encoded in the ‘functional linker’ are efficiently presented to CD8 T cells.
  • Human tumor cells were infected with VSV encoding for different TAME-IT molecules containing or not a ‘functional linker’ that contains antigenic peptides. These infected cells were then co-cultured or not with human monocyte-derived dendritic cells before being co-cultured for 6 hours with a human, HLA-A2 -restricted, CD8 T cell clone specific for the tumor antigen NY-ESO-1 which peptidic sequence is found in the ‘functional linker’.
  • T cell activation was evaluated by intracellular staining of gamma-interferon and was only observed against HLA-A2 + tumor cells and dendritic cells in the conditions where a VSV coding for TAME-IT molecules containing a ‘functional linker’ was used. This shows that the antigenic peptides from the TAME-IT molecules are efficiently processed by infected cells or immune cells and presented to tumor-associated antigen-specific human CD8 T cells.
  • TAME-IT proteins activate different types of human lymphocytes.
  • PD-L1 + human melanoma cells were co-cultured for 5 days with activated or non-activated human polyclonal CD8 T cells at target: effector ratios of 1 :2, 1:1 or 2: 1, in presence or not of the anti-PD-Ll antibody nivolumab or the TAME-IT protein KN035-TAA-th4-lBBL.
  • the confluence of the target melanoma cells was analyzed by microscopy during the experiment to evaluate killing by CD8 T cells.
  • the TAME-IT protein enhances the activation of CD8 T cells and the killing of melanoma cells.
  • MDA-MB-231 human breast tumor cells were infected for 18h with a VSV encoding or not for the KN035-TAA-th4-lBBL TAME-IT molecule. These infected cells were then co-cultured for 6h with human natural killer (NIC) cells. NIC cell activation was evaluated by surface staining of CD 107a and was increased in the condition where the VSV coding for the TAME-IT molecule was used.
  • FIG. 8 KN41 induces greater activation of lymphocytes T.
  • A Total PBMCs were co-cultured with Ml 13 or M113-PD-L1 cells (10,000 tumor cells per well) at the indicated target: effector ratios in the presence or absence of beads coupled to anti-CD3/CD28 antibodies at a ratio one ball for 20 PBMC. The cells were either treated simultaneously with KN41 or pre incubated with KN41 (KN41 PI). After 24 h, the supernatants were harvested and assayed for IFNg by ELISA.
  • B Under the conditions with 5 PBMC per tumor cell and anti-CD3/CD28 beads, the IFNg in the supernatant was assayed at the indicated times.
  • LentiX cells (HEK-293T) were plated at 7.10 5 cells per well in a 6-well plate at day 0. At day 1, the medium was replaced by 2mL of fresh DMEM complemented with 10% FCS. The cells were transfected using Lipofectamine 3000 with 2pg of pcDNA3.1 plasmid coding for the TAME-IT proteins. 48h post-transfection, the supernatants were collected and cleared by centrifugation at 10,000g for 10 minutes.
  • Mesol3 human mesothelioma cells or HEK-293T cells were incubated with the supernatants from control (EFGP) or TAME-IT -transfected HEK cells for 30 minutes. After 2 PBS washes, cells were stained with anti-h4-lBBL or anti-hCD40L antibodies (Biolegend, cat#311504 and 310806, respectively) for 15 minutes. After 2 PBS washes, cells were analyzed using a BD Accuri Cl flow cytometer.
  • LentiX cells (HEK-293T) were plated at 7.10 5 cells per well in a 6-well plate at day 0. At day 1, the medium was replaced by 2mL of fresh DMEM complemented with 10% FCS. The cells were transfected using lipofectamine 3000 with 2pg of pcDNA3.1 plasmid coding for our constructions. 48h post-transfection, the supernatants were collected and cleared by centrifugation at 10,000g for 10 minutes.
  • DC Dendritic cell
  • Human monocytes from healthy donors were plated at 2.10 6 cell per mL and 1.10 6 cells per cm 2 in 6-well plates in RPMI-1640 medium supplemented with 2% human albumin, granulocyte-macrophage colony-stimulating factor (GM-CSF, 500 IU/mL) and Interleukin-4 (IL-4, 50 ng/mL). After 5 days of culture (37°C, 5% C02), the differentiated DCs were collected and counted.
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • IL-4 Interleukin-4
  • 100,000 differentiated DCs were incubated for 48h in a 96-well plate, either directly with the supernatants from HEK cells containing our molecules, or with tumor cells previously stained with these supernatants and washed before the co-culture.
  • Monocyte-derived DCs were collected by flushing at the end of the incubation time. After 2 PBS washes, they were stained with anti-hCD80, anti-hCD83, anti-hCD86 and anti- hHLA-DR antibodies for 30 minutes. After 2 PBS washes, cells were analyzed using a BD FACS Canto II flow cytometer. CD80, CD83 and CD86 expression was determined on the HLA-DR+ cell fraction corresponding to DCs.
  • TAME-IT molecules can be vectorized by being encoded in an oncolytic virus (OV) and efficiently produced by OV-infected cells.
  • OV oncolytic virus
  • VSV Vesicular Stomatitis Virus
  • MVA-T7 Vesicular Stomatitis Virus
  • VSV-L-coding vectors VSV-N, VSV-P, VSV-L-coding vectors and the anti-genomic VSV vector containing the TAME-IT sequence.
  • Recombinant VSV were recovered from the supernatants by 0,22pm filtration and several additional passages on BHK-21 cells.
  • the genes encoding for the TAME-IT proteins were inserted in the genome of an oncolytic strain of Vaccinia Virus by homologous recombination.
  • Cells were collected by flushing at the end of the incubation time. After 2 PBS washes, they were stained with an anti-hCD3 antibody for 30 minutes. After 2 PBS washes, cells were fixed in 4% paraformaldehyde, permeabilized with PBS + 0.1% saponine and stained with an anti-hlFNy antibody for 30 minutes. After 2 PBS + 0.1% saponine washes, cells were analyzed using a BD Canto II flow cytometer. T cells were gated as CD3+ cells and analyzed for intracellular IFNy production.
  • a first experiment using CD4 T cell clones shows that TAME-IT proteins containing a ‘functional linker’ are efficiently produced by tumor cells infected with a VSV encoding them (Figure 5). These molecules are further processed and presented on MHC class II molecules by DCs having previously phagocytosed VSV-TAME-IT-infected tumor cells. These DCs subsequently activate TAA-specific CD4 T cells. This demonstrates that the ‘functional linker’ allows the specific activation of CD4 T cells that recognize antigenic peptides encoded in this linker. These results were confirmed in two additional experiments with DCs from different donors (data not shown).
  • TAA peptides from the ‘functional linker’ are also efficiently processed and presented on MHC class I molecules to TAA-specific CD8 T cells by (i) tumor cells infected with VSV encoding for TAME-IT proteins and (ii) DCs having previously phagocytosed VSV-TAME-IT-infected tumor cells (Figure 6). These results were confirmed in two additional experiments with DCs from different donors (data not shown).
  • PD-L1 + human melanoma cells were seeded in 96-well plate at a concentration of 10,000 cells per well.
  • human polyclonal CD8 T cells were added at ratios 2:1, 1:1, 1 :2 onto tumor cells with or without activation beads previously coated with anti-CD3 and anti- CD28.
  • the anti-PDl antibody nivolumab or the purified TAME-IT protein KN035-TAA-th4-lBBL were added in the mix. The confluence of tumor cells was analyzed for 5 days by time-lapse microscopy.
  • TAME-IT proteins have the property to activate different types of human T lymphocytes in the context of anti -tumour immunity.

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Abstract

Des protéines chimériques pour l'immunothérapie du cancer peuvent associer différentes activités en affichant à la fois des propriétés agonistes et antagonistes sur une seule molécule. Cependant, celles-ci présentent généralement des toxicités liées au fait que leur puissance s'exerce dans l'ensemble de l'organisme et pas seulement dans les tissus concernés par le traitement du cancer. Ceci peut être résolu en utilisant une vectorisation appropriée des protéines chimériques dans le microenvironnement tumoral, où les cellules immunitaires interagissent avec des cellules tumorales exprimant des molécules immunomodulatrices. La présente invention concerne un acide nucléique codant pour une protéine chimérique ou une protéine chimérique ayant la structure générale suivante : N terminal - (a) - (b) - (c) - C terminal, dans laquelle : (a) représente un domaine de signalisation et/ou de ciblage, (b) représente un lieur, (b) représente un lieur fonctionnel et (c) représente un domaine de signalisation et/ou de ciblage, ou N terminal - (c) - (b) - (a) - C terminal, dans laquelle : (c) représente un domaine de signalisation et/ou de ciblage, (b) représente un lieur, (b) représente un lieur fonctionnel et (a) représente un domaine de signalisation et/ou de ciblage.
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